Methods and devices to enhance sensitivity and evaluate sample adequacy and reagent reactivity in rapid lateral flow immunoassays

ABSTRACT

Methods and devices for rapid lateral flow immunoassays to detect specific antibodies within a liquid sample while also validating the adequacy of the liquid sample for the presence of immunoglobulin and the integrity and immunoreactivity of the test reagents that detect the antibodies of interest, without requiring instrumentation. The methods and devices provide for delivery of a diluted liquid sample to a single location that simultaneously directs the liquid flow along two or more separate flow paths, one that serves as a positive control to confirm that all critical reagents of the test are immunoreactive, and that the sample being tested is adequate, and the other to detect specific antibodies if present.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Patent Application No.61/258,074, filed Nov. 4, 2009, expressly incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to immunoassay devices and the methodsfor their use. More specifically, the invention relates to rapidimmunoassays that utilize capillary lateral flow for rapid detection ofligands within biologic fluids. The invention relates to methods anddevices that permit evaluation of sample adequacy and confirmation ofcritical reagent immunoreactivity during performance of rapid lateralflow immunoassays, and to methods and devices that provide increasedsensitivity within such immunoassays.

The methods and devices specifically pertain to confirmation of adequatelevels of immunoglobulin in the liquid sample being evaluated in therapid lateral flow immunoassay, and to confirmation of immunoreactivityof antigens, particulate markers, and any control monoclonal antibodies,polyclonal antibodies or other ligands that are present on the membranesof the assay. The methods and devices also specifically provide simpledevice designs and procedures that allow ligands to interact with theirspecific binding partners more favorably than in conventional lateralflow rapid diagnostic tests with respect to interference from ordetection by particulate markers. The present invention provides rapidtests with higher sensitivity than conventional lateral flowimmunoassays.

The invention methods and devices are illustrated using HIV antibodiesand antigens, and human C-Reactive Protein and its ligands. Thesemethods and devices of the invention provide a platform strategy anddevice procedures applicable to rapid detection of antibodies specificto other antigens, or for detecting other ligand binding pairs and othermedical conditions.

BACKGROUND OF THE INVENTION

Many rapid lateral flow diagnostic tests are in use throughout the worldtoday. Rosenstein was the first to describe the methodology (U.S. Pat.No. 5,591,645) in 1987, and many others have contributed improvementssince then (U.S. Pat. No. 4,855,240, Rosenstein et al.; U.S. Pat. No.5,602,040, May et al.; U.S. Pat. No. 5,714,389, Charlton et al.; U.S.Pat. No. 5,824,268, Bernstein et al.; and U.S. Pat. No. 7,189,522,Esfandiari). However, none of these tests that detect antibodies tospecific antigens provide a means within each test to simultaneouslydetect whether the sample being evaluated actually contains a sufficientamount of immunoglobulin to allow detection of specific antibodies, ifpresent. Also, none of these tests currently available have built-incontrols to simultaneously report immunoreactivity of the criticalreagents required for the test to provide accurate results. These twoimportant omissions mean that some tests may provide falsely negativeresults due to inadequate sample or failed reagents. When a person hasHIV antibodies, and they provide an inadequate sample to the rapid testfor evaluation, or when the critical reagents of the test havedeteriorated, no lines will develop that would normally indicate apositive result, such as antibodies in the test sample to HIV antigen onthe test membranes. The test user is likely to incorrectly conclude thatthe test is negative because no lines have appeared. However, hadcontrols for sample adequacy and reagent reactivity been included in thetest, this mistake would not occur. The user would observe that eitherthe sample was inadequate for testing, or that critical reagentsrequired for accurate test performance were not working, and make thecorrect conclusion that the test result was INVALID and could not beinterpreted, rather than falsely concluding that the test was negative.

Specific examples of this type of failure of rapid tests for HIV havebeen reported in Kenya and Uganda (Mar. 28, 2009—Uganda Monitor Online,Dr. Z. Akol, the STD/AIDS Control Programme manager at the HealthMinistry, Kampala, Uganda) and noted failures of tests given to 6,255people due to exposure to harsh storage conditions and using testsbeyond their expiration dates, failures that would be detected withcontrols for antigen and critical reagent reactivity, but not without,and also failures due to errors the tests short reading windows of 20minutes or less, reading beyond which may give false positives. InIndia, more than one hundred thousand persons were tested in five stateswith faulty tests that lacked controls to detect reagent failure orsample inadequacy—76,464 persons were tested in Mumbai alone, in theperiod 2-07 through 5-07, and S Kudalkar, chief of the Mumbai DistrictAIDS Control Society (MDACS) stated “It is those who tested negative inthose three months who are a cause for worry. Since they aren't testedagain, if their result is wrong, they could have missed appropriatetreatment.” Dr. A. Deshpande also pointed out that pregnant mothers withHIV infection may have been missed. The Delhi High Court ruled on May25, 2009 that all those tested between February-07 and May-07 must beretested due to the poor quality of the HIV rapid tests used (M.Rajadhyaksha, TNN 29 Jul. 2009).

Many current rapid lateral flow tests contain a so-called positivecontrol, which is nothing more than a incomplete procedural control thatsimply reports that fluid introduced into the rapid test has migrated tothe end of the test results window (see U.S. Pat. No. 5,989,921,Charlton et al., claim 1, line 26, “said control site comprising animmobilized binder that binds said conjugate,” regardless of whetherantibody is present or absent in the sample. Also see U.S. Pat. No.5,656,503, May et al., claim 42 (d), line 63 “a control zone downstreamfrom said test result zone in said dry porous carrier for bindinglabeled reagent to indicate that said applied liquid biological samplehas been conveyed by capillarity beyond said test result zone.” Again,the binding is independent of the presence or absence of the analyte ofinterest in the liquid biological sample. Also see OraSureOraquickADVANCE Rapid HIV-1/2 test PMA, that refers to “a goat anti-human IgGprocedural control immobilized onto a nitrocellulose membrane in the . .. . Control (C) zone” . . . “This built-in procedural control serves todemonstrate that a specimen was added to the vial and that the fluid hasmigrated adequately through the test device”. These controls do notcatch false negative results that may result from testing an inadequatesample or failure of critical test reagents.

Current advanced instrument based tests for detection of antibodies toHIV typically will detect the presence of antibodies to HIV a few dayssooner during seroconversion following infection than can be detectedwith rapid lateral flow diagnostic tests, and well before Western Blotconfirmatory tests become positive. It would be desirable for rapidlateral flow diagnostic tests to have increased sensitivity that allowedthem to more closely approximate the sensitivity of instrument basedtests.

Current rapid lateral flow diagnostic tests for HIV are approved by theUS FDA for use with only very short reading windows, beyond which thetest must not be interpreted due to an increased risk of false positiveresults. For example, the Trinity Biotech UniGold Rapid HIV-1 antibodytest must be read over a 2 minute window, precisely 10-12 minutes aftertest initiation. The Inverness Determine HIV-1/2 antibody test must beread over a 5 minute window between 15 and 20 minutes, and theOraSureOraQuick ADVANCE HIV-1/2 test must be read over a 20 minutewindow between 20 and 40 minutes. In busy emergency room or urgent careclinics, this limited reading period for the rapid tests can bedifficult to achieve, resulting in either failure to read the testappropriately, or failure of widespread use of the test due to itslimitations. It would be desirable to have a test with a reading windowof at least several hours, and preferably several days to months.

Currently existent rapid lateral flow diagnostic test devicesoccasionally permit backflow of test reagents from the downstreamabsorbent pad, upstream into the reading window. When this occurs, thetest results are obscured and accurate reading of the results becomesimpossible. It would be desirable to have a method and device thatutilized the method that prevents such backflow of reagents.

SUMMARY OF THE INVENTION

An important object of this invention provides methods, and rapidlateral flow immunoassay devices that utilize the methods, that decreasethe chances that an erroneous result will occur when untrained usersutilize the improved methods and devices of this invention as comparedto conventional lateral flow rapid immunoassays.

It is therefore an object of this invention to provide a method, andrapid lateral flow immunoassay device that utilizes the method, toevaluate sample adequacy in terms of whether the sample containssufficient immunoglobulin to allow detection of immunoglobulin moleculeswithin the sample that are specifically directed at antigens or analytesof interest.

It is a further object of the invention to provide a method, and rapidlateral flow immunoassay device that utilizes the method, to confirmreactivity of all critical reagents of the rapid test. This includesconfirmation of reactivity of immobilized antigens or analytes, of anypositive control mobilizable immunoglobulins utilized within theimmunoassay format, and reactivity of the particulate marker thatdetects immunoglobulin molecules within the biological liquid samplethat specifically recognize the immobilized antigens or analytes ofinterest, and that also recognizes positive control immunoglobulinswithin the test format.

An additional particularly desirable object of the invention is toprovide a method, and a rapid lateral flow immunoassay device thatutilizes the method, that allows oral fluid to be collected from thetooth-gingival crevice line by a custom swab that fits directly into thedevice and permits direct dilution of that gingival crevice fluid fromthe swab and analysis of that diluted sample for specific antibodies.Since many patients prefer tests that do not require a blood sample, theability to test oral fluid in a simple to perform test should providewider acceptability for its use.

It is also an object of the invention to provide a method, and a rapidlateral flow immunoassay device that utilizes the method, that providesincreased the sensitivity of the immunoassay as compared to conventionallateral flow immunoassays, by providing more favorable interactionsbetween specific binding pairs included within the assay format.

It is an additional object of the invention to provide a method, and arapid lateral flow immunoassay device that utilizes the method, thatmaintains the simplicity of performance and interpretation inherent inrapid lateral flow immunoassays, that has permitted worldwide use ofsuch rapid tests by untrained users.

It is a further object of the invention to provide a method, and a rapidlateral flow immunoassay device that utilizes the method, that increasesthe reading window over which the test result can be accuratelyinterpreted from between 10 to 15 minutes at the earliest, to up to 24hours or more.

It is also an object of the invention to provide a method, and a rapidlateral flow immunoassay device that utilizes the method, that preventsbackflow of reagents from the downstream absorbent pad of the rapid testdevice into the upstream reading window for the test.

An additional object of the invention is that any test devices thatutilize the methods of this invention be designed as compatible withinjection molding to produce the plastic housing parts for the device,and that the designs are suitable for automated assembly manufacturing.

As discussed in detail in the following Examples, Figures and DetailedDescription, this invention has developed methods, and devices thatachieve the above objectives.

This invention has discovered a combination of specific immunoglobulinbinding reagents that allow detection of the presence of immunoglobulinin rapid lateral flow formats, while overcoming problems with thenon-specific interactions that occur with many binding pairs whichprevent accurate assessment of whether adequate immunoglobulin ispresent in test samples.

The invention further provides methods and devices that allowconfirmation of immunoreactivity of the antigen being used to detectantibodies specific to it, while at the same time confirming theimmunoreactivity of the particulate marker being used within the test,as well as immunoreactivity of any monoclonal antibodies or polyclonalantibodies being used to bind to critical epitope determinants of theantigen being used in the test to detect antibodies in the liquid samplebeing detected. This is accomplished by the test devices and membraneconfigurations provided in the invention that direct the liquid samplebeing tested along two or more separate flow paths.

One flow path, designated the positive control reagent pathway isspecifically designed to evaluate sample adequacy and theimmunoreactivity of the critical test reagents. It contains antigen orantigens immobilized on nitrocellulose in the downstream portion of thisflow path that is (are) identical to the same antigen or antigens coatedon the second flow path designed to detect antibodies specific to thatantigen or those antigens. The upstream portion of the positive controlreagent pathway contains dried mobilizable monoclonal or polyclonalantibodies or other ligands that bind specifically to the epitope orepitopes being recognized on the downstream test antigen or antigens,the same epitopes being recognized in the other flow path (or paths)over which liquid samples are evaluated for the presence of antibodyspecific to this antigen. The upstream portion of the positive controlreagent flow path also contains mobilizable dried particulate marker,that when mobilized will recognize and bind to the mobilizable driedmonoclonal or polyclonal antibodies or other ligands that specificallyrecognize the downstream test antigen or antigens When mobilized andmigrated over the downstream immobilized test antigen or antigens thebinding of the particulate marker to an immobilized antigen confirmsthree features: (1) immunoreactivity of the antigen, (2)immunoreactivity of the monoclonal or polyclonal antibodies directed atthe specific antigen, and (3) immunoreactivity of the particulatemarker. The positive control reagent pathway also contains furtherdownstream an immunoglobulin recognizing reagent that specificallyrecognizes immunoglobulin in the test sample if it is present insufficient amounts to allow accurate performance of the rapid test. Thisreagent is adjusted in concentration so that it does not bind sufficientpositive control antibodies to allow visually recognizable binding bythe mobilizable particulate marker of the positive control reagentpathway, but will show a visible line of binding of particulate markerif the diluted sample presented to the test device contains sufficientimmunoglobulin to be an adequate sample.

The second flow path (and any additional flow paths) is (are) designatedthe specific antibody flow path(s) for the purpose of detecting specificantibodies to immobilized antigens (analytes) of interest.

One advantage of the methods and devices provided in this invention isthat the positive control reagent pathway provides one or moreobservable lines indicating for each observable line that the antigenrecognized is reactive and that a comparable line can be expected forthat antigen in the specific antibody pathway if specific antibodies arepresent. The antigen used in each pathway is identical, and it isimmobilized using identical conditions in each pathway. Also,particulate marker used in the positive control reagent pathway isidentical to the particulate marker used in the pathway to detectantibodies to specific antigens. This provides assurance when no line ispresent in the specific antibody pathway that the negative result wasnot due to inactivity of the antigen or degradation of the particulatemarker. This type of control is intuitively easier to understand andfacilitates proper use and evaluation of the test results, as comparedto previously described controls in which a positive control is theabsence of a line. The positive control reagent pathway of the inventionis also preferable to controls in which the antigen is detected byimmobilized monoclonal antibody, because that type of control requiresthe antigen to be bound to particulate marker which means it isdifferent from native antigen used in the test, or if native antigen isused, it must be bound upstream to a monoclonal antibody or other ligandthat contains a particulate marker, and it has thus been modified and isnot directly comparable to the antigen used in the pathway for detectingantibodies specific to the test antigen.

The devices of this invention provide for the fluid being tested to bedirected into two or more separate pathways. Separating the positivecontrol reagent pathway that confirms reactivity of critical reagentsfrom a different pathway to detect antibodies specific to antigens ofinterest prevents interference of different control and test componentsthat would occur if positive control monoclonal or polyclonal antibodyreagents were combined into the pathway to detect test sample antibodiesto a specific antigen. The flow direction, from the membrane thatcollects the diluted liquid sample within the device into the two ormore separate pathways and ultimately into absorbent pads at thedownstream end of each pathway, prevents backward flow contamination ofthe specific antibody detection pathway by reagents found in thepositive control critical reagent pathway.

The invention includes the possibility of two or more separate ports,one allowing addition of buffer to the critical reagent positive controlpathway, and one or more additional ports to accept liquid sample beingtested for antibodies or ligands specific to immobilized antigens oranalytes of interest. This also would provide a means within thecontemplated invention to provide proof that the critical reagents ofthe test are intact, at the same time as evaluating the test results forthe presence or absence of antibodies to specific antigens or analytesof interest. However, the preferred embodiment of this invention, forpurposes of ease of use of the test, is to be able to add the liquidsample to a single location, and have the devices direct the liquidsample over two or more pathways from that single location, to provideobservable results from each pathway, as provided for in this invention.

It should be noted that the two or more lateral flow paths described inthis invention, are to be distinguished from the two paths or dual pathsfirst described by Rosenstein et al. (U.S. Pat. No. 4,855,240) andsubsequently by Bernstein et al. (U.S. Pat. No. 5,824,268) andEsfandiari (U.S. Pat. No. 7,189,522 B2). In each of these U.S. patents,particulate tracer is run over one pathway and the sample runs over aseparate pathway. In contrast, in this invention, both sample andparticulate marker are run over each pathway. This invention containscontrols to test for sample adequacy and for immunoreactivity of thecritical reagents whereas these three previously cited patents do notcontain these controls.

It should also be realized that U.S. Pat. No. 6,627,459 B1, entitled“Immunoassay Controls” by Tung et al is different from than the presentinvention. It contains a positive control that is non-cross reactivewith the biological analyte to be detected, whereas this invention usesthe identical antigen for the positive control which is used to detectspecific analytes, and both are identically immobilized onnitrocellulose. This is accomplished by placing the antigen positivecontrol in a separate pathway from the pathway to detect antibodiesspecific to that antigen, and simultaneously directing the sample beingtested over each pathway. In this way, the antigen immobilized and usedas the positive control does not interfere with the same antigen used inthe separate pathway which detects antibodies specific to that antigen.Also because the positive control antigen is stored under identicalconditions to the same antigen used in the pathway for detectingspecific antibodies, it is a preferable measure of possible adverseeffects on immunoreactivity of storage conditions, such as excess heat,excess humidity, exposure to oxygen, and storage beyond expirationdates, than a non-identical, non-cross reactive positive control asdescribed by Tung et al.

The invention provides a custom swab design that when utilized with therapid test device design, provides for isolation of a portion of swabmembrane saturated with gingival crevice fluid, which is then removedand simultaneously diluted and delivered to the test device according tothe method of Buchanan (U.S. Pat. No. 7,364,914 B2). The simplicity ofthis device design decreases the chance of user error. Furtheradvantages regarding oral fluid processing, capillary flow and speed oftest completion, and protection against spillage, are provided in thisinvention by collecting the diluted oral fluid sample into a membranerather than a well, and providing fluid communication from the dilutedsample membrane into the positive reagent control pathway as well as thespecific antibody pathway.

The invention also provides a designed insert that fits into the rapidtest device and allows separation of acellular components from two dropsof whole blood. The whole blood from a fingerstick or venipuncture mayeasily be added directly to the insert. Migration by capillarity withinthe insert separates acellular components of the blood at the leadingedge of the migration from trailing cellular components. A definedamount of this acellular portion is then isolated and diluted within thetest device and the diluted sample is directed into the flow pathways ofthe device for detection of antibodies to specific antigens.

This invention provides for more favorable interactions betweenmobilizable particulate marker, specific antibodies, and the immobilizedantigens to which those antibodies are directed as the result of twodesign changes. First, when the sample is diluted and removed from thesaturated swab or whole blood membrane by applying test running bufferunder pressure in the test device it is delivered to the center of thediluted sample membrane. The concentration of diluted sample along themembrane varies from most concentrated at the membrane periphery to moredilute as it approaches the center of the membrane, and finally nosample and only running buffer at the center of the diluted samplemembrane. The rapid test designs of this invention connect the peripheryof the diluted sample membrane with the upstream portion of the separatepathways; the reagent positive control pathway and the specific antibodypathway or pathways. Second, the test designs of this invention thatprovide increased sensitivity orient a strip containing particulatemarker so that its ends overlap the space between the periphery of thediluted sample membrane and the upstream portion of each pathway, butone side of the diluted sample membrane partially overlaps one side ofthe remaining particulate marker membrane. This orientation results inthe initial interaction of diluted sample and particulate marker beingidentical to conventional lateral flow tests. Diluted sample flowingdown each pathway pushes particulate marker ahead of it, some of whichdoes not interact with sample, and flows into the absorbent pad. Theleading edge portion of marker-sample interaction involves an excess ofimmunoglobulin relative to marker, necessitating dilution of the testsample in many antibody tests in order to produce observable results.The ensuing interactions of conventional lateral flow immunoassayscontain continued high levels of immunoglobulin relative to diminishingand then absent marker. In contrast, the new methods of utilizing newdesigns and orientation of particulate marker pads of this invention,provide continued supply of marker throughout test development. Thisinnovation, together with a diluted sample membrane that provides arange of dilutions of immunoglobulin to marker produce more favorablemarker-immunoglobulin complexes that produce observable results.Together, these more favorable characteristics result in highersensitivity for detection of ligands of interest in the diluted sample.

The new generation design of this invention also increases theproportion of complexes that are capable of producing observable bindingof particulate marker to immobilized antigens by providing continueddelivery of particulate marker to the rapid immunoassay test over a muchlonger time course of the test. Antibodies that have flowed down eachpathway and bound to antigens prior to complexing with marker that wouldhave been missed with conventional lateral flow techniques may berecognized by this new generation design that provides subsequent markerflowing down the same pathways. Unfavorable ratios of marker to antibodybinding that produced complexes too large to flow paths smoothly alongthe test device pathways also have an opportunity to reach morefavorable size and flow conditions by continued flow past them of markerexcess or antibody excess solutions during different periods of therapid test design of this invention. Finally, the new generation testdesign provides running buffer that contains no antibody or particulatemarker in the final stages of flow of fluid through the rapid testpathways. This final running buffer wash facilitates removal of anyresidual particulate marker from the reading window of the rapid testthat has not specifically bound to immobilized antigens or ligands,providing a white background against which to easily read the testresults.

The invention maintains the simplicity of lateral flow immunoassays thatmakes it easy to use and interpret. In the oral fluid version, the usersimply swabs the gingival crevice margin with the provided custom swaband then places it into its precise location in the test device. Thedilution port is then pushed down to lock in place and isolate a sectionof swab membrane saturated with oral fluid and the tip of the providedvial of running buffer is inserted into the opening in the top of thedilution port and squeezed to force out the liquid simultaneouslyremoving and diluting the oral fluid from the isolated membrane anddelivering it into the test device. The test result can then be readthrough the windows of the test device in approximately ten minutes. Inthe whole blood version, an insert to collect whole blood is placed intothe opening of the test device cover in the precise location thatreceives the swab in the oral version. This insert contains a membranethat isolates acellular from cellular blood components when two drops ofblood from a fingerstick or venipuncture are placed into the funnelreceptacle of the insert, and allowed to migrate for 4 minutes from thereceiving area to the opposite end of the sample receiving membrane. Thedilution port is then pushed down into the locked position and runningbuffer is added from the provided vial as in the oral fluid test and theresult may be observed in the reading windows of the test device after 6additional minutes.

This invention includes design modifications that provide a largeabsorbent pad that absorbs all of the one ml of diluted sample fluidadded to the test device. This large volume absorbent pad, plus designof the outflow wicks from the downstream ends of the reagent positivecontrol pathway and specific antibody pathway connecting to theabsorbent pad and the device design that includes overlying vent areasin the cover of the test device to facilitate evaporation of test liquidfrom the absorbent [pad and keep] pad, keep all flow within the testdevice from upstream to downstream portions of each pathway and into theabsorbent pad, and prevent backflow into the reading window that mightlimit the reading time over which the test result may be accuratelyread.

The plastic components of the test device and the orientation of themembranes within the device are designed to be compatible with automatedassembly manufacturing.

The following is a description of a representative method of theinvention with reference to the flow paths and reference numerals setforth in FIG. 13. In one embodiment, the invention provides a method fordetecting an analyte in a liquid sample, comprising:

(a) isolating a portion of the compressible membrane containing at leasta portion of a liquid sample to be analyzed by applying compressiveforce on top and bottom surfaces of the membrane along the perimeter ofan area to be isolated thereby defining a non-compressed area of themembrane centripetal to the compressed perimeter;

(b) delivering a liquid diluent under pressure to the isolated portionof the membrane to release at least a portion of the liquid sample fromthe isolated portion of the membrane to provide a diluted sample;

(c) contacting the diluted sample with a second porous membrane (8)having a first end and a second end, wherein the diluted sample flowstoward the first and second ends (FD2);

(d) conducting the diluted sample from the first end of the secondporous membrane to the first end of a third porous membrane (upper 7)having a first end and a second end, wherein the third porous membranecomprises a mobilizable marker that binds to the analyte and amobilizable analyte binding partner (e.g., F240), and wherein thediluted sample further comprises the marker and analyte binding partneronce the diluted sample flows to the second end of the third porousmembrane;

(e) conducting the diluted sample from the second end of the secondporous membrane (8) to the first end of a fourth porous membrane (lower7) having a first end and a second end, wherein the fourth porousmembrane comprises the mobilizable marker and wherein the diluted samplefurther comprises the marker once the diluted sample flows to the secondend of the fourth porous membrane;

(f) conducting the diluted sample further comprising the marker from thesecond end of the third porous membrane to the first end of a fifthporous membrane (9) having a first end and a second end, wherein thefifth porous membrane comprises a first region comprising an immobilizedanalyte binding ligand (e.g., HIV antigen) wherein the immobilizedanalyte binding ligand is effective in binding the mobilizable analytebinding partner (and the analyte, e.g., HIV antibody), and the marker iseffective in binding to the mobilizable analyte binding partner (and theanalyte) (bound to the immobilized ligand), as the diluted samplecomprising the marker and mobilizable analyte binding partner flowstoward the second end of the fifth porous membrane, thereby providing anindication that the mobilized binding partner, immobilized analytebinding ligand, and marker are operating satisfactorily; and

(g) conducting the diluted sample further comprising the marker from thesecond end of the fourth porous membrane to the first end of a sixthporous membrane (10) having a first end and a second end, wherein thesixth porous membrane comprises a first region comprising theimmobilized analyte binding ligand wherein the immobilized analytebinding ligand is effective in binding the analyte and the marker iseffective in binding to the analyte (bound to the immobilized ligand) asthe diluted sample comprising the marker flows toward the second end ofthe fifth porous membrane, thereby providing an indication that thesample includes the analyte, when the analyte is present in the sample.

In one embodiment, the method further comprises conducting the remainingdiluted sample from the second end of the fifth porous membrane to thefirst end of a seventh porous membrane (upper 11) having first andsecond ends, wherein the seventh porous membrane draws the dilutedsample from the second porous membrane (FD3); and conducting theremaining diluted sample from the second end of the sixth porousmembrane to the first end of an eighth porous membrane (lower 11) havingfirst and second ends, wherein the eighth porous membrane draws thediluted sample from the second porous membrane.

In one embodiment, the fifth porous membrane (9) further comprises asecond region comprising an immunoglobulin binding partner immobilizedtherein (for example, downstream from the immobilized ligand), whereinthe immunoglobulin binding partner is effective in binding theimmunoglobulin in the liquid sample, and wherein the mobilizable markeris effective for binding to the immunoglobulin, as the diluted sampleflows toward the second end of the sixth porous membrane, therebyproviding an indication that the sample has immunoglobulin sufficient toconfirm sample adequacy.

In one embodiment, the sixth porous membrane (10) further comprises asecond region comprising an immunoglobulin binding partner immobilizedtherein (for example, downstream from the immobilized ligand), whereinthe immunoglobulin binding partner is effective in binding theimmunoglobulin in the liquid sample, and wherein the mobilizable markeris effective for binding to the immunoglobulin, as the diluted sampleflows toward the second end of the sixth porous membrane, therebyproviding an indication that the sample has immunoglobulin sufficient toconfirm sample adequacy.

In one embodiment, the liquid sample is added to the compressible porousmembrane to provide a portion of the compressible membrane containing atleast a portion of the liquid sample.

In one embodiment, the liquid sample is whole blood. In one embodiment,the liquid sample is gingival crevice oral fluid.

In one embodiment, the analyte binding partner is an antigen and theanalyte is an antibody. In one embodiment, the analyte binding partneris an antibody and the analyte is an antigen. In one embodiment, theanalyte binding partner is the HIV antigen and the analyte is the HIVantibody.

The following is a description of a representative device of theinvention with reference to the flow paths and reference numerals setforth in FIG. 13. In one embodiment, the present invention provides adevice for detecting an analyte in a liquid sample, comprising:

(a) a compressible porous membrane for receiving a liquid sample;

(b) first and second members adjacent opposing major surfaces of thecompressible porous membrane, wherein the first and second members areengageable to apply compressive force on top and bottom surfaces of themembrane along the perimeter of the area to be isolated, therebydefining a non-compressed area of saturated membrane centripetal to thecompressed perimeter;

(c) a port adapted to receive delivery of a second fluid, wherein theport is further adapted to deliver the second fluid under pressure tothe isolated portion of the membrane to force the removal of at least aportion of the liquid sample from the isolated portion of the membraneto provided a diluted sample;

(d) a second porous membrane (8) for receiving diluted liquid samplefrom the compressible membrane, the second porous membrane having firstand second ends;

(e) a third porous membrane (upper 7) comprising a mobilizable markereffective in binding to the analyte and a mobilizable antigen bindingpartner (e.g., F240), the third porous membrane having first and secondends, wherein the first end of the second porous membrane is in liquidcommunication with the first end of the third porous membrane;

(f) a fourth porous membrane (lower 7) comprising the mobilizablemarker, the fourth porous membrane having a first end and a second end,wherein the second end of the second porous membrane is in liquidcommunication with the first end of the fourth membrane;

(g) a fifth porous membrane (9) comprising a first region comprising animmobilized analyte binding ligand (e.g., HIV antigen), wherein theimmobilized analyte binding ligand is effective in binding themobilizable antigen binding partner (and the analyte), the fifth porousmembrane having a first end and a second end, wherein the second end ofthe third porous membrane is in liquid communication with the first endof the fifth membrane;

(h) a sixth porous membrane (10) comprising a first region comprising animmobilized analyte binding ligand (e.g., HIV antigen), wherein theimmobilized analyte binding ligand is effective in binding the analyte,the sixth porous membrane having a first end and a second end, whereinthe second end of the fourth porous membrane is in liquid communicationwith the first end of the sixth membrane.

In one embodiment, the device further comprises a seventh porousmembrane (upper 11) for drawing the diluted sample from the secondporous membrane (FD3), the seventh porous membrane having a first endand a second end, wherein the second end of the fifth porous membrane isin liquid communication with the first end of the seventh membrane; andan eighth porous membrane (lower 11) for drawing the diluted sample fromthe second porous membrane (FD3), the eighth porous membrane having afirst end and a second end, wherein the second end of the sixth porousmembrane is in liquid communication with the first end of the eighthmembrane.

In one embodiment, the fifth porous membrane further comprises a secondregion comprising an immunoglobulin binding partner immobilized therein(for example, downstream from the immobilized ligand), wherein theimmunoglobulin binding partner is effective in binding theimmunoglobulin in the liquid sample, and wherein the mobilizable markeris effective for binding to the immunoglobulin, as the diluted sampleflows toward the second end of the fifth porous membrane, therebyproviding an indication that the sample has immunoglobulin sufficient toconfirm sample adequacy.

In another embodiment the sixth porous membrane further comprises asecond region comprising an immunoglobulin binding partner immobilizedtherein (for example, downstream from the immobilized ligand), whereinthe immunoglobulin binding partner is effective in binding theimmunoglobulin in the liquid sample, and wherein the mobilizable markeris effective for binding to the immunoglobulin, as the diluted sampleflows toward the second end of the fifth porous membrane, therebyproviding an indication that the sample has immunoglobulin sufficient toconfirm sample adequacy.

In one embodiment, the first end of the second porous membrane overlapsthe first end of the third porous membrane. In one embodiment, thesecond end of the third porous membrane overlaps the first end of thefifth porous membrane. In one embodiment, the second end of the fifthporous membrane overlaps the first end of the seventh porous membrane.In one embodiment, the second end of the second porous membrane overlapsthe first end of the fourth porous membrane. In one embodiment, thesecond end of the fourth porous membrane overlaps the first end of thesixth porous membrane. In one embodiment, the second end of the sixthporous membrane overlaps the first end of the eighth porous membrane.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings.

FIG. 1 is an exploded perspective view of a representative device of theinvention including a yoke (1), a sample dilution port (2), a cover (3),a swab with suitable absorptive fabric and of correct dimensions forcollecting oral fluid and fitting into the device (4), an o-ring (5), amid-piece (6), a diluted sample membrane (7), two or more membranescontaining particulate marker conjugate and in the case of the controllane a reagent that binds to the conjugate and recognizes the downstreamantigen or antibody (8), one or more test membranes (9), a controlmembrane (10), absorbent pad membranes (11) and a base (12).

FIG. 2A is a top plan view, FIG. 2B is a front elevation view, FIG. 2Cis a bottom plan view of sample dilution port (2).

FIG. 3A is a cross section view of dilution port (2, FIG. 1) through itscentral channel (36, FIGS. 3A and 10C) and its hook arms (32, FIG. 2B)and FIG. 3B is a cross section view of dilution port (2) through one ofits hook arms (32). The plane of the cross section and the direction ofsight for FIG. 3A are indicated by the dotted line and arrows which arelabeled A in FIG. 2A. The plane of the cross section and the directionof sight for FIG. 3B is indicated by the dotted line and arrows labeledB in FIG. 2A.

FIG. 4A is a top plan view and FIG. 4B is a front elevation view of yoke(1). Yoke has arm surfaces (13) that hold it within the dilution portand finger tabs (14) which allow it to be withdrawn from the dilutionport when the arm surfaces (13) are compressed toward each other.

FIG. 5A is a top plan view of the cover (3) for the oral fluid rapidtest device. It illustrates windows to view test (16) and control (17)results, evaporation ports (19) for test and control strips, and anopening to receive the sample dilution port, two edges of which containprojecting ridges (18) which mate with corresponding projecting ridges(35, FIG. 2B) of the sample dilution port when it is in the depressedand locked position (see FIGS. 9A and 9E). FIGS. 5B and 5C are front andperspective views of the cover respectively, that illustrate the opening(20) for the oral fluid swab that contains the oral fluid to be sampledduring performance of the oral fluid rapid test.

FIG. 6A is a top plan view of the oral fluid swab, with dotted lines toshow the area of fabric on the swab to be sampled (21) by the sampledilution port (FIGS. 10B and 10C) when the swab containing collectedoral fluid is placed into the device opening for same (FIGS. 5B, 5C, and20). FIG. 6B is a right side view of oral fluid swab that illustratestapering of the swab in the region containing the fabric that collectsoral fluid. This tapering provides the correct dimensions to allowcompression and isolation of a portion of the fabric on the end of theswab by the dilution port to allow production of a diluted sample oforal fluid to the device during testing.

FIGS. 7A, 7B, and 7C are top plan, perspective, and front views of theoral fluid midpiece respectively. FIG. 7A contains ridges (24) thatposition the midpiece on base (FIG. 1, 12), a depression (23) to hold ano-ring, and a central channel (25) that permits flow of diluted samplefrom the top side to bottom side of the midpiece. FIGS. 7B and 7C showo-ring (26, also FIG. 1, 5) in place within its channel in the midpieceand the protruding exit area (27) for channel (25) on the bottom surfaceof midpiece.

FIGS. 8A, 8B, and 8C are top plan, perspective and front views of thebase (FIG. 1, 12). FIG. 8A illustrates placement of the diluted samplemembrane (8), test nitrocellulose membrane (9), control nitrocellulosemembrane (10) and absorbent pads (11) within the base. FIG. 8Billustrates the receptacles (29) within the base receive the hook arms(FIG. 9C, 32) of the dilution port which enter the receptacle throughopening 28 (FIG. 8A) of the receptacles. FIG. 8C shows windows 30 in thereceptacles through which the hook arms of the dilution port protrudeonce the dilution port is pushed down into the receptacles until thehook arms (32, FIG. 2; 32S, FIGS. 9D and 9E) lock beneath ridge 33(FIGS. 8B and 9E). This protrusion into window 30 of the receptacle bythe hook arms locks the dilution port in place in four separate areas,one protruding hook arm into each window 30 of each receptacle.

FIG. 9 further illustrates how the dilution port (2) inserts into thebase (12). FIG. 9A is a top plan view of the cover (3) with dilutionport (2) and yoke (1) in place. The dotted line in FIG. 9A is the planeof a cross-sectional view through the middle of one of the hook arms 32(FIG. 9C) to produce FIGS. 9D and 9E, and the line of sight for thesefigures is illustrated by the arrows. FIG. 9D shows cross sections ofthe hook arms relative to the base receptacles in the up-unlockedposition and FIG. 9E shows the hook arms in the down-locked positionwithin the base receptacles. FIG. 9B illustrates how the yoke (1)prevents the dilution port (2) from being depressed down into the coveropening by passing through openings (34, FIGS. 2B and 10B). It may beremoved by pushing the yoke arms (13, FIGS. 4B and 13, FIG. 9B) towardeach other while simultaneously pulling on finger tabs (14, FIG. 4A).Once the yoke is removed the dilution port may be depressed to thelocked position within the base receptacles, and its top surface is thenflush with the top surface of the opening for the dilution port in thecover (FIG. 9C, 2). Once flush with the cover the ridges (35, FIG. 2B)on the undersurface of the periphery of the long dimension of thedilution port cover abut ridges (18, FIG. 5A) of the cover. Theseprevent the cover from being removed once the dilution port is lockedinto the base. FIG. 9D is a cross-sectional left view of a hook arm inits base receptacle in the up position. Hook arms (32S) sit above theupper ridges (33S) of the base receptacle. FIG. 9E is a cross-sectionalleft view of a hook arm in its base receptacle in the down and lockedposition. The locking of dilution port into the receptacle isaccomplished by four points, two for each hook arm, as illustrated byhook arms 32S now being located beneath ridges 33S which prevent thedilution port from moving back upward once locked into position.

FIG. 10 illustrates how an area of swab fabric once saturated with oralgingival crevice fluid is isolated and used to produce the dilutedsample analyzed by the test device. FIG. 10A is a top plan view of thecover with dilution port in place. The dotted line indicates the planeof the cross-sectional view of the dilution port through the top openingof the central channel (31, FIG. 9A) and the line of site for viewingcross sections in this plane for FIGS. 10B and 10C is indicated by thearrows and the letters B and C. FIG. 10B is the cross section with thedilution port in the up and unlocked position, and FIG. 10C is the crosssection with the dilution port in the down and locked position. FIG. 10Bshows openings (34) in the dilution port (3S) from which the yoke wasremoved to allow the dilution port to be depressed into the device.Other components of the dilution port in the FIG. 10B cross sectioninclude the central stem of each hook arm (35S), the central channel(36) of the dilution port through which diluent passes after beingintroduced through the opening in the top of the dilution port (31,FIGS. 10B and 9A), and receptacles (29S) in the base (12S) that acceptthe hook arms of the dilution port. FIG. 10B illustrates cross sectionsof the inferior surfaces (37S) of the walls of the dilution portsurrounding its central channel which together with an o-ring (5S)located within a channel in the midpiece (FIG. 7A, channel 23, and FIG.7B, o-ring 5) provide compressive force on top and bottom surfaces,respectively, of the swab fabric (FIG. 10B, 21S) saturated with oralgingival crevice fluid in order to isolate an area of non-compressedsaturated membrane.

FIG. 11 is an enlargement of FIG. 10C. It shows cross sectional area ofcompression of the swab membrane, and also illustrates the location ofthe diluted sample membrane (8S) relative to the midpiece. FIG. 11 showsthe isolated area of non-compressed swab fabric (26) which is locatedcentripetal to the perimeter of compression formed by o-ring (FIG. 10B,5S) and inferior surfaces of the dilution port (FIG. 10B, 37S). Whendiluent fluid is applied under pressure in the direction of the arrowthrough channel 36, FIG. 10C, it removes the isolated sample of gingivalcrevice fluid from the isolated area of non-compressed membrane (26S,FIGS. 10C and 11) and dilutes it. The diluted sample exits from beneaththe midpiece through a channel formed by inferior projections (27S, FIG.11) on its undersurface and into the diluted sample membrane (FIG. 11,8S and FIG. 1, 8) and from there wicks onto the other membranes of thetest device. FIG. 11 also demonstrates a cross section of the frame(38S) of the oral swab. It occupies the periphery of the portion of theoral swab that collects oral fluid, leaving a central area into whichthe dilution port may be depressed (FIGS. 10C and 11) to isolate theportion of saturated swab fabric to be used in the test.

FIG. 12 illustrates the position of membranes of the test devices inrelation to the midpiece. FIG. 12A is a bottom plan view showing theplacement of particulate marker pads 7 in relation to the exit ofcentral channel 25 (FIG. 7A) from the bottom of midpiece through exitregion 27 of the midpiece (FIG. 7C). FIG. 12B is a bottom plan viewshowing the overlap region (dotted line) between the particulate markerpads 7 and the diluted sample membrane 8. FIG. 12C is a 2× right view ofmidpiece 6 showing the orientation of the diluted sample membrane 8 tothe particulate marker pads. When diluent fluid is introduced underpressure in flow direction 1 (FD1, FIG. 12C) through the isolatedmembrane 26 centripetal to o-ring 5, it removes sample from the isolatedmembrane and the diluted sample passes through the midpiece and exitsfrom the bottom of the midpiece where it comes into contact with dilutedsample membrane 8. From a central location on diluted sample membrane 8,it flows outward in flow direction 2 (FD2, 12C) to come into contactwith particulate marker pads 7 for each flowpath. FIG. 12D is a 2× frontview of the device that demonstrates the overlap between diluted samplemembrane 8 and particulate marker pad 7, between particulate marker pad7 and nitrocellulose membranes 9 or 10, and between nitrocellulosemembranes 9 or 10 and absorbent pads 11. Flow direction 3 (FD3, FIG.12D) for the diluted sample is from diluted sample membrane 8 up andacross particulate marker membrane 7, downward from particulate marker 7into nitrocellulose membrane 9 or 10, across these nitrocellulosemembranes and finally upwards again into absorbent pads 11 at thedownstream end of each nitrocellulose membrane.

FIG. 13 further illustrates the flow directions of running buffer anddiluted liquid sample during use of the device to detect specificantibodies or analytes in oral fluid. In this figure, the midpiece andbase are removed and only membranes of the test are displayed, each inits correct orientation as it exists within the medical devices of thisapplication. The sample membrane (21) sits on top of the midpiece (notshown in this figure, see 6, FIG. 12) and once the sample membrane hasbeen saturated with the liquid to be tested, a defined area of thatsaturated sample membrane (26) is isolated according to U.S. Pat. No.7,364,914, B2, Buchanan. The isolated area of saturated membrane iscentripetal to a perimeter of compression of the membrane formed by ano-ring within the midpiece (5, FIG. 12) pressing on the undersurface ofthe membrane, and opposing matching inferior surfaces of the dilutionport (37, FIGS. 2B and 2C, and 37S, FIG. 10B) compressing the topsurface of the membrane. Flow Direction 1 (FD1, FIGS. 11, 12, and 13)occurs when test running buffer is delivered under pressure through thecentral channel of the dilution port (36, FIG. 10C), through theisolated portion of the saturated membrane containing the sample (26S,FIG. 11), through channel 25 within the midpiece (FIG. 7A) and out theinferior opening of the midpiece located beneath the isolated membrane(27S, FIGS. 10C and 27, FIGS. 12A, 12D). The sample is removed from themembrane and diluted and dropped onto the diluted sample membrane (8,FIGS. 1, 8A, 12B-D, 13, and 8S, FIG. 11). Entry of the diluted sample inthe diluted sample membrane initiates flow direction 2 (FD2, FIGS. 12Cand 13) which transfers the fluid from the central portion of thismembrane to its periphery which allows contact with the particulatemarker pads located on the undersurface of the midpiece (7, FIGS. 1,12A-D, 13) to initiate flow direction 3. Flow direction 3 (FD3, FIGS.12D and 13) occurs in two or more flow paths, one to confirmimmunoreactivity of all critical test reagents (results displayed onmembrane 10 (FIGS. 1,8& 13) through cover window 17 (FIGS. 5A and 9A)and an additional one or more paths to detect specific antibodies ifpresent and also confirm sample adequacy (results displayed on membrane9 (FIGS. 1, 8, and 13) through cover window 16 (FIGS. 5A and 9A). Flowdirection 3 transfers the diluted liquid sample by capillary flow fluidcommunication and membrane wicking from the upstream portion of eachflow path which includes mobilizable dried reagents on the particulatemarker pads (7, FIGS. 1, 12A-D and 13) into the downstream portion ofeach flow path that consists of a nitrocellulose membrane containingimmobilized antigen and an additional reagent to evaluate sampleadequacy in the sample adequacy and specific antibody detection flowpath. The final downstream portion of each flow path is an absorbent padthat collects all the excess running buffer and any unbound componentsof the diluted sample and mobilized reagents used in the test. Theabsorptive capacity of this pad dictates that fluid added to the dilutedsample membrane (8, FIGS. 1,8A, 12B-D, 13) will end up in the absorbentpad and that the overall test flow directions will be upstream todownstream along a path of diluted sample membrane leading toparticulate marker pad to nitrocellulose membrane and finally to theabsorbent pad.

FIG. 14 shows top plan views of the test device cover. Results that mayoccur with the rapid lateral flow test methods and devices of thisinvention are shown in FIGS. 14A-14D. The detailed description andinterpretation of these results is found in Example 7.

FIG. 14A shows absence of a line demonstrating binding of particulatemarker to immobilized antigen in window 17 of the cover, over thereagent control pathway. This indicates failure of critical testreagents. There is no line of binding to antigen in the specificantibody and sample adequacy pathway viewed through window 16 of thecover, and a line exists further downstream to indicate that the sampletested contained adequate immunoglobulin for evaluation.

FIG. 14B shows a line demonstrating binding of particulate marker toimmobilized antigen in window 17 of the cover, over the reagent controlpathway. This indicates that the critical test reagents are working.Window 16 over the pathway to test sample adequacy and to test forantibody to specific antigens shows no line of binding to specificantigen and also fails to show binding of particulate marker the Igbinder rPA immobilized further downstream in the same path. The lack ofbinding to immobilized rPA indicates that the sample tested containedinsufficient immunoglobulin for evaluation.

FIG. 14C shows a line of particulate marker binding to immobilizedantigen in window 17 of the cover, over the reagent control pathway, andalso shows in window 16 of the cover a line of particulate markerbinding to the Ig binding reagent rPA immobilized downstream in thesample adequacy pathway. These lines indicate that sample is adequatefor testing and that all critical reagents are working. There is no lineof binding of particulate marker to antigen in the specific antibody andsample adequacy pathway viewed through window 16 of the cover.

FIG. 14D shows a line of particulate marker binding to immobilizedantigen in window 17 of the cover, over the reagent control pathway, andalso shows in window 16 of the cover a line of particulate markerbinding to the Ig binding reagent rPA immobilized downstream in thesample adequacy pathway. These lines indicate that sample is adequatefor testing and that all critical reagents are working. There is a lineof binding of particulate marker to antigen in the specific antibody andsample adequacy pathway viewed through window 16 of the cover.

FIG. 15A is a top plan view of a revised cover device design for usewith modifications that provide improved test sensitivity, longerreading windows and lack of backflow of test reagents. It contains alarger opening (43) for the dilution port (2, FIG. 1), increasedventilation areas (39) to speed evaporation of test running buffer anddiluted sample, tapered windows (40 and 41) to assist reading andinterpreting results for the positive reagent and sample adequacypathway (window 40) and the specific antibody detection pathway (window41), and an indented area (42) into which may be placed a protectivepolyester cover for the test device strips upon which may be printeddesired labeling to assist identification and interpretation of testresults.

FIGS. 15B-15E illustrate representative results that may be obtainedusing the device designs of this invention. In these figures, thereagent reactivity and sample adequacy pathway results are shown inwindow 40 with rounded edges, and the specific antibody detectionresults are shown in rectangular window 41.

FIG. 15B shows a line to indicate sufficient immunoglobulin in thereagent positive control and sample adequacy pathway, but no line ofparticulate marker over the immobilized antigen. This indicates failureof the immobilized antigen, the particulate marker or the upstreammobilizable monoclonal antibody to the immobilized antigen of interestand the test result must be interpreted as INVALID.

FIG. 15C shows a line of particulate marker over the immobilized antigenin the reagent positive control and sample adequacy pathway, whichindicates that antigen, particulate marker, and monoclonal antibody tothe immobilized antigen are immunoreactive. However, there is no line ofparticulate marker over the immobilized recombinant protein A. Thisindicates failure that the sample being tested contained insufficientamounts of immunoglobulin to allow detection of antibodies specific tothe immobilized antigen to be detected if present, and the test resultmust be interpreted as INVALID.

FIG. 15D shows lines of particulate marker binding over the immobilizedrecombinant protein A (44) and over the immobilized antigen (45) as readthrough the window overlying the reagent positive control and sampleadequacy pathway. This indicates that the test is functioning properlyand the absence of any line of particulate marker binding to immobilizedantigen in the rectangular window over the specific antibody pathwayindicates that the diluted sample contains no antibodies specific to theimmobilized antigen and the test result may be correctly interpreted asNEGATIVE.

FIG. 15E shows lines of particulate marker binding over the immobilizedrecombinant protein A and over the immobilized antigen in the reagentpositive control and sample adequacy pathway. This indicates that thetest is functioning properly and the presence of a line of particulatemarker binding to immobilized antigen in the rectangular window over thespecific antibody pathway indicates that the diluted sample containsantibodies specific to the immobilized antigen and the test result maybe correctly interpreted as POSITIVE.

FIG. 16A is a top plan view of a revised base design that provides alarge well (67) capable of holding an absorbent pad that can hold 1.1 mlof liquid (50, FIG. 16B). This large absorbent pad reservoir ensuresthat fluid flow direction is from the upstream point of entry into eachflow path, flowing downstream over each flow path and terminating inthis pad, and that backflow of pad contents into the viewing windows ofthe test device is prevented as described in Example 9. The spacing ofthe most central base receptacle (68) for the dilution port that hasbeen moved in the revised base 3 mm closer to the absorbent pad well(67). This provides 3 mm of extra space (49) on the downstream side ofdiluted sample well (48) that is outside of and adjacent to the dilutedsample well and allows use of a modified particulate marker pad (55,FIG. 16B) with resulting increased test sensitivity as described inExamples 10 and 11.

FIG. 16B is a top plan view of the revised base design with membranes ofthe rapid test in place, except for bridging membranes that connect thenitrocellulose strip of control (52) and test (53) membranes across gap(54) between them and a particulate marker pad (55) that has beenmodified to perform tests with increased sensitivity as described inExamples 10 and 11. The particulate marker pad (55) sits predominantlyon a plastic shelf adjacent the diluted sample membrane (56) and itsupstream edge overlaps the diluted sample membrane by 1-2 mm. Thediluted sample membrane is held within a well designed for it in thebase (48, FIG. 16A). Nitrocellulose membranes of the reagent positivecontrol and sample adequacy pathway (52) and specific antibody pathway(53) overlap on their downstream with outflow wicks (51) connecting thenitrocellulose strips to the absorbent pad (50).

FIG. 16C is similar to FIG. 16B except that bridging membranes (7, FIG.12) represented by dashed lines 59 in FIGS. 16C and 16D cross gap 54(FIG. 16C) between upstream diluted sample membrane 56 and downstreamnitrocellulose membranes when the midpiece (60, FIG. 16D) is placed intothe device so that bridging membranes (59, FIG. 16D) are positioned asshown. These bridging membranes allow fluid communication betweenparticulate marker pad 55 (FIG. 16C) and nitrocellulose membrane ofcontrol pathway 52 (FIG. 16B) and specific antibody pathway 53 (FIG.16B) when wet by diluted sample during test performance.

FIG. 16D shows the revised base of FIG. 16A with the membranes of FIG.16B in place along with midpiece 60 of FIG. 16C with bridging membranes(dashed lines 59, FIGS. 16C and 16D) attached to the undersurface of themidpiece. FIG. 16D also shows o-ring 23 located within its groove in theupper surface of midpiece 60. This o-ring facilitates isolation of anarea of saturated sample membrane centripetal to the o-ring when themembrane is compressed on its undersurface by the o-ring and on its topsurface by the bottom of the dilution port as described in U.S. Pat. No.7,364,914 B2, Buchanan.

Once diluted sample is created within the test device during testperformance as described in Examples 5 and 6, the fluid migrates bycapillarity through the membranes from upstream diluted sample membrane56 to downstream absorbent pad 50 as shown by dashed lines 58 of FIG.16C. The dashed circle 57 of FIGS. 16B, C, E, and F shows the area onthe undersurface of the midpiece from which the diluted sample entersthe diluted sample membrane.

FIG. 16E illustrates the flow patterns (dashed lines 58) for movement ofdiluted sample into two flow paths, each with a different size andorientation of the particulate marker membrane. Flow path 52 is thesample adequacy and positive reagent reactivity control pathway and inthis illustration the particulate marker pad (68, FIG. 16E) is ofconventional size and orientation design. The conventional particulatemarker pad for this pathway is the same width as the width of thedownstream pathway membranes. The flow path 53 (FIG. 16E) for detectionof specific antibody contains a new generation particulate marker pad(67, FIG. 16E) that overlaps the diluted sample membrane along one edgeand allows a greater range of concentrations of sample to interact withparticulate marker as explained in Example 10 and FIG. 16F, and alsoprovides particulate marker to the developing lateral flow immunoassayover a longer time period during test result development (Example 10).Together, these changes result in higher sensitivity of the rapidimmunoassay as compared to conventional lateral flow rapid test designs(Example 11).

FIG. 16F illustrates how particulate marker pads of the invention (67,FIG. 16F), are oriented to the diluted sample membrane (56, FIG. 16F)with a slight overlap. The diluted sample concentrations obtained by thedevices of the invention are preserved within the membrane in a rangefrom highest concentrations near the ends of the membrane to lowestconcentrations near the entry point to the membrane from the midpieceisolated sample (57, FIGS. 16B, D, E, and F, and Example 10). Thisoverlap provides a relatively constant amount of particulate marker tointeract with sample dilutions ranging from as low as 0-10% to as highas 85-100%. During test development with the invention, the firstmarker-sample interactions to enter the downstream flow path are thosewith highest concentrations of sample (FIG. 16F). Next, sampleconcentrations 40-70% of those that initially entered the downstreampath, that have been incubating with particulate marker in a higherratio of marker to sample arrive at the upper end of the specificantibody flow path and migrate downstream (FIG. 16F). Finally,combinations with the highest ratios of marker to sample (constantmarker combined with sample at 10-40% of most concentrated, see Example10) reach the entry to the specific antibody flow path and flow over itsdownstream components.

In contrast to the varied concentrations of sample to marker tested withthe invention, the particulate marker pad of conventional design (68,FIG. 11F) interacts only with the highest concentrations of dilutedsample in the range of 70-100% of highest concentrations, and thereafterno particulate marker is available to interact with diluted sample as itmigrates down the flow pathway (pathway 52, FIG. 16F).

When conventional particulate marker pads (68, FIG. 11F) were comparedto the particulate marker pads of the invention (67, FIG. 11F), it wasfound that particulate marker was delivered to the assay 3 times longerwith the new design and orientation than with conventional in-lineparticulate marker pads (Example 10). The new designs and orientationsof particulate marker pads in this invention provided a 5 to 10 foldincrease in sensitivity for detection of specific antibody when comparedto conventional lateral flow immunoassays employing in-line marker pads(Example 11).

FIG. 17A is a 2D perspective view of the top portion of the insert, andFIG. 17B is a 2D perspective view of the bottom portion of the insert. Amembrane such as LF1 (GE Whatman, Florham Park, N.J., USA), cut to theprecise size of the inner margin of the insert bottom, sits on platform62 and is held in place within the insert by compression between the topand bottom portions of the insert. When 2 drops of fingerstick orvenipuncture whole blood are placed directly into funnel-like portion 61(FIG. 17A) of the test device insert, the blood flows onto the LF1membrane and then over a 4 minute period migrates to the opposite end ofthe membrane with acellular serum or plasma components preceding themore slowly migrating cellular components. A portion (66, FIG. 18) ofthe LF1 membrane saturated with acellular components of the whole bloodsample may then be isolated and diluted by the method of Buchanan (U.S.Pat. No. 7,364,914 B2).

FIG. 18 shows the assembled whole blood insert in place within therevised cover. When two drops of whole blood are introduced onto the LF1membrane at position 65, the acellular portion of blood migrates to andbeyond position 66 within 4 minutes. When the dilution port is pusheddown and locked into place, the area of LF1 membrane saturated withacellular-enriched whole blood is then isolated between the bottomsurface of the dilution port and the o-ring in the upper surface of themidpiece (FIG. 10C). When running buffer is then applied through thedilution port opening under pressure, the acellular-enriched whole bloodis simultaneously removed from the isolated portion of saturated LF1membrane and diluted and delivered to the diluted sample membrane (56,FIG. 16C and from there flows downstream from one end into the absorbentpad over the reagent positive control and sample adequacy pathway, andfrom the other end into the specific antibody pathway, as illustrated bydashed lines 58 of FIG. 16C.

DETAILED DESCRIPTION OF THE INVENTION

A specific description of how the methods and devices of thisapplication allow determination of sample adequacy is provided inExample 3 and Table 1 below. The basic approach was to discoverconditions of a rapid lateral flow immunoassay capable of providing aqualitative indication of the presence of immunoglobulin in the liquidsample being tested, the absence of which indicates that the sample isinadequate for detecting antibodies specific to a given antigen, sincethe specific antibodies of interest would be a sub-fraction of theimmunoglobulins detected by the sample adequacy test.

There are many potential user errors that might contribute to testing asample that contained inadequate amounts of immunoglobulin, and theabsence of a built-in control to detect this type of error might leadthe user to falsely conclude that the test result was negative, ratherthan realizing that the collected sample was inadequate. For example, inthe oral fluid test, instead of collecting oral fluid from the gingivalcrevice at the teeth-gum line, the user might simply collect saliva ontothe swab, or collect moisture from the mucosal lining of the cheeks.Alternatively, the location of collection might be correct but the timeinsufficient, such as collecting the sample in 3 seconds rather than 30seconds each over the teeth-gum line junction. These errors wouldproduce a fluid with far less immunoglobulin on the swab fabric than ispresent in a properly collected sample. For the whole blood test usingfinger-stick or venipuncture blood, the first portion of the assay ismigration of the blood along a membrane that slows migration of thecellular components and leaves an area of serum or plasma at the leadingedge of the saturated membrane that is then sampled by the dilution portto o-ring perimeter compression method to isolate a non-compressedportion of membrane saturated with serum or plasma for testing (seeAhlstrom CytoSep or GE-Whatman LF1 membranes, and U.S. Pat. No.7,364,914 B2). This initial migration along the blood sample collectingmembrane requires 4 minutes to provide a leading edge of serum or plasmacontaining depleted cellular components that is ready for dilution andtesting. If the user decides to depress the dilution port into thelocked position and pass running buffer through the locked dilution portto perform the test after waiting for only 3 minutes or less,insufficient migration will have occurred to provide an adequate sample.This will be detected by the sample adequacy control methods describedin Example 3 allowing the correct interpretation that the test isinvalid. The absence of such a control means that this type of usererror would not be detected and that the test result might be falselyinterpreted as negative, rather as an invalid test that requiresrepeating to obtain an answer.

In theory, many of the combinations presented in Table 1 wouldreasonably be expected to work for confirming the presence ofimmunoglobulin in the test samples. In practice however, under theconditions described herein and with membranes in common use in manylateral-flow immunoassays, it was observed that only one of the 13different combinations evaluated produced a line when immunoglobulin waspresent in the test sample, and no line when only the sample diluentwithout immunoglobulin was tested. Each of the 12 other combinationsproduced a line at the most downstream end of the test strip where theIgG binding reagent was immobilized on nitrocellulose even when no IgGwas present in test sample. This non-specific line was nearly equivalentto the line resulting from test samples that contained immunoglobulin.

The observation from these experiments was that non-specific bindingoccurs between most Ig binding reagents bound to colloidal gold whenexposed to other Ig binding reagents immobilized on nitrocellulosedownstream (12 examples). In contrast, when recombinant protein A(Repligen Corporation, Waltham, Mass.) was immobilized on nitrocelluloseit retained its ability to recognize IgG in samples containing IgG boundto protein L colloidal gold and protein G colloidal gold (BioAssayWorks, Ijamsville, Md.) when both of these colloidal gold reagentsmigrated past it from upstream to downstream, but failed tonon-specifically bind to a mixture of protein L colloidal gold andprotein G colloidal gold when they were migrated past the rPA in runningbuffer containing no IgG (Ig binding reagent line 26 of Table 1). Wheneither protein G or protein L colloidal gold preparations were migratedalone, in running buffer without IgG, past downstream immobilized rPA,they each bound non-specifically to the downstream immobilized rPA. Onlythe combination of BOTH protein L colloidal gold and protein G colloidalgold failed to bind non-specifically to low concentrations ofrecombinant protein A to produce a visibly detectable line. Theassumption from these results is that colloidal gold preparations ofprotein G and protein L together block some or most of the non-specificbinding sites recognized by immobilized rPA when either colloidal goldprotein G or colloidal gold protein L are migrated individually past theimmobilized rPA.

Rapid diagnostic tests may also fail to produce an accurate test resultif the critical reagents required to produce detection of antibodies tospecific antigens have deteriorated due to storage under adverseconditions or past the shelf-life expiration date. Reagent failure canresult in false negative results. Current tests do not have built-incontrols to test for this possibility. The two most critical componentsthat may fail are (a) antigens immobilized on nitrocellulose that mustcontain intact antigenic sites that are recognized by antibodiesspecific to those antigens as they migrate past and (b) Ig bindingreagents bound to particulate markers. If the immobilized antigen beingused to test for specific antibodies has deteriorated, antibodiesspecific to this antigen will simply migrate past it in the rapid assay.Without a control for antigen integrity, the lack of a line at the siteof immobilized antigen may be falsely interpreted as a negative test.Similarly, no line will result to indicate the presence of specificantibodies if the Ig binding reagents bound to particulate markers arenot immunoreactive. These particulate markers will fail to recognizespecific antibody in the test sample bound to immunoreactive immobilizedantigen, and migrate past. Again, this potentially may result in thefalse interpretation that no specific antibodies to the antigen beingtested are present in the test sample.

This invention provides built-in controls to confirm immunoreactivity ofthe critical immobilized antigen and of the particulate marker-Igbinding complexes, as well as the immunoreactivity of a third criticalreagent, a monoclonal antibody or polyclonal antibodies thatspecifically recognize the immobilized antigen for which specificantibodies are being detected. This is accomplished by having a pathwaydedicated to testing immunoreactivity of the tests critical reagentsthat is separate from the pathway used to detect antibodies specific toa given antigen, and by using the same antigen or antigens immobilizedidentically on the downstream portion of both the specific antibody andcritical reagent pathways, and by using the same particulate marker-Igbinding reagent complexes dried down identically in mobilizable formonto the particulate marker pads on the upstream portion of bothpathways. In addition, the critical reagent pathway contains amobilizable reagent or reagents, dried onto the upstream portion of thepathway that bind(s) to the specific antigen or antigens immobilizeddownstream. This or these reagents may be monoclonal or polyclonalantibodies or other molecules that specifically recognize the antigen towhich specific antibodies are being detected in the separate specificantibody and sample adequacy pathway, and are also recognizable by theIg binding reagents bound to particulate markers. Preferentially, thispositive control antibody must be stable for as long or longer than theimmobilized antigen and the particulate markers bound to Ig bindingreagents must also preferentially be stable for as long or longer thanthe immobilized antigen, when dried in mobilizable form onto theupstream portion the critical reagent pathway. More preferentially themonoclonal antibody or polyclonal antibodies will recognize the samespecific antigenic determinants of the immobilized antigen that arerecognized by antibodies in the test samples being evaluated. Theadvantage of having the critical reagent pathway separated from thepathway to detect antibodies to specific antigens is that the antigenrecognizing control antibodies could interfere with detection ofimmobilized antigen by antibodies in the diluted test samples if theywere in the same pathway. With two separate pathways, the flowdirections of the rapid test (FIG. 13) prevent contamination of thespecific antibody pathway reagents with positive control antibodies fromthe critical reagent pathway.

Example 1 provides specifics of the preparation and use of potent andstable peptide-BSA conjugate antigen that is useful to detect antibodiesto HIV.

Example 2 provides specifics of how two monoclonal antibodies, one to alinear epitope and the other to a conformational epitope in the sameregion of gp41 of HIV-1, have been used in the critical reagent positivecontrol pathway to confirm immunoreactivity of immobilized peptide-BSAconjugate antigen used to detect human antibodies to HIV (U.S. Pat. No.5,260,189, (Formoso, Olsen, and Buchanan). It also provides a specificexample of the use of polyclonal serum antibodies as a positive controlfor the methods and devices used in this invention, for rapid lateralflow immunoassay of antibodies to human CRP.

Example 3 presents specifics and summary data on the evaluation of 12combinations of reagents that did not prove useful for confirming thepresence or absence of immunoglobulin in test samples. It also describesthe discovery of a single binding reagent combination that was able todistinguish samples that contained human immunoglobulin from those thatdid not under the conditions used in the methods and devices of thisinvention.

Example 4 provides specific conditions used to identify a suitablefabric for the collection of gingival crevice oral fluid samples, torelease fluid from the fabric and to identify HIV specific antibodieswithin the diluted gingival crevice fluid using the test strips andreagents of this invention.

Example 5 provides specific details of the use of the oral fluid rapidtest device as described in the figures to detect antibodies to HIV inhuman oral gingival crevice fluid.

Example 6 provides specific details of the use of one whole blood rapidtest device of this invention for detection of antibodies to HIV infingerstick or venipuncture whole blood.

Example 7 and FIG. 14 provide illustrations of results obtained with useof the rapid test platform of this invention. Shown are a True Positive,a True Negative, and two Invalid tests. One invalid was falsely Negativedue to inactive critical reagents, and the other Invalid test wasfalsely Negative due to insufficient immunoglobulins in the test sample.

Example 8 describes a study demonstrating that monoclonal antibody F240is sufficiently potent that very small quantities are required to bindenough of the monoclonal to allow recognition by particulate markerprotein G+L colloidal gold and produce a line of particulate binding tothe immobilized HIV-1 antigen, leaving insufficient monoclonalantibodies to migrate past the immobilized antigen to be recognized bydownstream immobilized recombinant protein A and cause a line ofparticulate marker at the downstream location. This allows all controls(both reagent reactivity and sample adequacy) to be placed on the samepathway and simplifies interpretation of the test results.

Example 9 and FIGS. 16C-16E provide specific details of a test designwicking system and large absorbent pad sink located at the downstreamend of the test device that allow capture of all of the liquid added tothe test device after it has passed through test and control strips,while simultaneously preventing backflow of test reagents from absorbentpad to test and control strips. Currently available lateral flow testshave limited reading windows during which test results may be reliablyevaluated, due at least in part to backflow of reagents from theabsorbent pad into the area where the test result is evaluated. Backflowprevention is accomplished in this invention by both the size of theabsorbent pad which is capable of absorbing the total liquid added tothe test device, and the location of evaporation vents in the testdevice that are oriented over the absorbent pad and the outflow wicksthat move liquid from the downstream end of test and control strips intothe absorbent pad. After all liquid added to the test device has flowedinto the absorbent pad, evaporation first results in drying of thesurface of the absorbent pad and the overlying wick that connects thedownstream end of the test and control strips and absorbent pad. Anyliquid remaining in the partially evaporated absorbent pad remains inthe inferior aspect of the pad, unable to complete backflow into testand control strips due to evaporation while traversing the dried surfaceof the absorbent pad and downstream end of the dried wick. Using thisdesign, no backflow of reagents from absorbent pad to test or controlstrips occurred during more than one week of observation.

Example 10 explains the details of methods and design innovations in (a)the size and orientation of particulate marker pad membranes of thisinvention, that when combined with (b) methods and designs that permitinteraction of a range of sample concentrations with marker during thetest, as illustrated in FIGS. 16B, C, E, and F.

Example 11 provides details of experiments that demonstrate that theinnovations described in Example 10 together provide increasedsensitivity for the detection of known quantities of monoclonal antibodyspecific to HIV-1 antigen as described in Examples 1 and 2 (Table 2,Example 11), and for detection of seroconversion from HIV negativity topositivity, as compared to conventional lateral flow immunoassays (Table3, Example 11). These changes suggest an approximate 5 to 10 foldincrease in sensitivity using the methods and design innovations of thisinvention.

Example 12 confirms the importance of importance of diluting the testsample sufficiently to not overwhelm the amount of particulate markeravailable to combine with immunoglobulin.

Dilutions of 1:200 or greater of human serum appear to be optimal forthe amount of particulate marker available in rapid tests using themethods and designs of this invention (Table 4, Example 12). FIG. 11Fillustrates how the methods and designs of this invention allowimmunoglobulin interaction with particulate marker not only in the sameconcentrations as conventional lateral flow immunoassays, but also inamounts 10 to 100 fold lower. This 10 to 100 fold range of availableconcentrations of immunoglobulin that interact with a constant amount ofparticulate marker with the methods and designs of this invention,increase chances for optimal ratios that allow maximum sensitivity. Whentime periods were compared for delivery of particulate marker to thedeveloping immunoassay with conventional particulate marker pads (68,FIG. 16F) as compared to the newly designed and oriented particulatemarker pads of this invention (67, FIG. 16F), particulate marker wasdelivered to the developing assay over a three times longer period thanwith conventional lateral flow immunoassays (Table 5, Example 12).

Example 13 and FIGS. 17A, 17B, and FIG. 18 describe the use and designof an insert that fits precisely with correct orientation into theopening in the cover of the test devices of this invention, where theswab would otherwise be placed when testing oral samples, the may beused to evaluate whole blood. It contains a funnel like portion in itstop that is designed to receive two free-falling drops of whole blood,that then migrate along the membrane of the insert to enrich thedownstream component of the whole blood for acellular components. After4 minutes, the dilution port of the test device (2, FIG. 1) is pusheddown into the locked position to isolate an area of membrane saturatedwith acellular enriched whole blood which is then removed and diluted bydiluent applied under pressure according to U.S. Pat. No. 7,364,914 B2)and diluted sample is delivered through the opening on the underside ofthe midpiece (FIG. 12) into the diluted sample membrane at area 57(FIGS. 16B, C, E, and F) to flow to the pathways illustrated in FIG. 13and FIGS. 16 C, E, and F where the results are read through the windowof the cover as illustrated in FIGS. 15B-15E.

FIGS. 15B-15E illustrate INVALID, TRUE POSITIVE and TRUE NEGATIVE testresults viewed through the test result windows of the revised cover ofFIG. 15, with tests performed as in Examples 5-7 in which the sampleadequacy recombinant protein A control is immobilized downstream on thesame nitrocellulose strip as the reagent positive control pathway.

Use of a Method and Device for Collection and Testing of Oral FluidSamples

The oral fluid collection device of this invention, as illustrated inFIGS. 1-16, is used as follows:

Using the swab provided (4, FIG. 4; and FIGS. 6A and 6B) the user rubsone side of the swab up and down along the entire upper and lowertooth-gum lines.

The swab is then inserted into opening 20 of the test device (FIG. 5).The dilution port 2 (FIGS. 1, 2 and 3) is then pushed down into thelocked position (FIGS. 9E, 10C and 11) after first removing the yoke (1,FIG. 1) that prevents premature depressing and locking. This locking isaccomplished by 4 separate contact points, two on each of two hook armsof the dilution port (32, FIG. 2B) that snap out into window 30 of thebase receptacles 29 for the dilution port hook arms (FIGS. 8A-C) andabut against the upper edge 33 of each window (FIG. 8B, and 33S, FIG.9E) thereby preventing the hook arms from being withdrawn from thedevice once depressed and locked. This locked position of the dilutionport isolates a portion of the swab fabric which has been saturated withoral gingival crevice fluid, by a perimeter of compression around theisolated fabric caused by an o-ring (5, FIGS. 1, 7B and 7C, and 5S, FIG.11) on the undersurface of the fabric and an opposing pressure from amating inferior surface of the dilution port (37, FIGS. 2C and 37S,FIGS. 11 and 12). Also, once the dilution port is depressed in to thelocked position, the cover of the test device cannot be removed becausethe swab (FIG. 6) locked in place by the dilution port (FIG. 2B) whichis locked into the base prevents removal of the cover to access thedilution port in order to unlock it. This design prevents attempts tore-use the test device.

A vial or syringe provided with a known quantity of running buffer isinserted into opening 31 (FIG. 9A) of the dilution port to form aleak-proof friction fit, and the buffer is pushed out of the vial orsyringe under pressure and down through central channel 36 of thedilution port (FIG. 10C) through the isolated saturated membrane therebyremoving sample which travels through channel 25 of the midpiece (FIG.7A) to exit through the channel 27 on the bottom surface of the midpiece(FIG. 7C) providing a diluted sample to the diluted sample membrane (8,FIGS. 12B-12D and FIG. 13). No more than 15 seconds are required forthis entire process after insertion of the oral swab into the device anddepression of the dilution port until completion of delivery of runningbuffer from the provided vial or syringe.

The diluted sample travels through the diluted sample membrane in flowdirection 2 (FD2, FIG. 13), and from there in flow direction 3 (FD3,FIG. 13) through upstream and downstream portions of the two or morepathways of the test, to provide readable results in ordinarycircumstances within 10 minutes.

The upstream portion of the reagent positive control and sample adequacypathway contains dried mobilizable monoclonal or polyclonal antibodiesto the antigen being used to detect specific antibodies, located on thebridging membrane between it and the particulate marker pad, and furtherupstream mobilizable particulate markers such as colloidal gold ormagnetic spheres bound to protein L and protein G, that recognize boththe mobilized monoclonal or polyclonal antibodies and humanimmunoglobulins. As the monoclonal or polyclonal antibodies aremobilized by sample diluted with running buffer and containing mobilizedparticulate markers, some migrate ahead of the particulate marker andbind to downstream immobilized antigen prior to interacting withparticulate marker and others interact with particulate marker as theymigrate toward immobilized antigen. A line is formed at the site ofantigen immobilized on nitrocellulose or other suitable membrane locateddownstream (on membrane 10, FIG. 13). The formation of this lineconfirms particulate markers bound to protein L and protein G arereactive, that the monoclonal or polyclonal antibodies directed at theimmobilized antigen are reactive and that the immobilized antigen inthis positive reagent control flow pathway is also immunoreactive. Thisline may be observed through window 40 in the cover of the device (FIG.15A) at location 45 of FIG. 15D. Absence of this particulate marker lineat the immobilized antigen can result from failure of any of the threecritical positive control pathway reagents. This result of noparticulate marker binding to antigen means that the test must beinterpreted as invalid by the user, thereby preventing potentiallyreporting a false negative result due to failure of any one or more ofthe three critical test reagents. Further down this same pathwayimmunoglobulins within the diluted sample that have complexed withparticulate marker are recognized and bound by recombinant protein A(Repligen Corp., Waltham, Mass., USA, 44, FIG. 15D). Absence of a lineat this location means that the sample being tested does not containsufficient immunoglobulin to allow detection of antibodies to specificimmobilized antigens if they are present in the diluted sample.

The upstream portion of the pathway for detection of specific antibodiescontains the same dried mobilizable particulate markers complexed withprotein G and protein L as found in the positive control reagentpathway. The downstream portion of this pathway contains identicalantigen immobilized with identical positioning on nitrocellulose orother suitable membrane under conditions identical to those used forthis antigen to coat on identical downstream membranes in the positivecontrol reagent and sample adequacy pathway. Observation of no line ofparticulate marker binding to the immobilized antigen in this pathwaythrough window 41 of the cover (FIG. 15A) at position 46 (FIG. 15E), butdefinite lines of identical particulate marker bound to antigen andrecombinant protein A in the reagent positive control and sampleadequacy pathway, allows the conclusion that the diluted test sample istruly negative for antibodies specific to the immobilized antigen. Thepresence of a line of particulate marker bound to immobilized antigen inthe specific antibody pathway, along with a line over immobilizedidentical antigen and a line over immobilized recombinant protein A inthe reagent positive control and sample adequacy pathway, allows theconclusion that the diluted oral fluid sample is truly positive forantibodies specific to the immobilized antigen.

Use of a Method and Device for Collection and Testing of Whole BloodSamples

The principles of the methods of this invention, as described above inthe section on use of the oral fluid testing device, are identical fortesting both whole blood and oral samples. The differences are found inthe test device.

In the newly designed device for testing whole blood from fingersticksor venipuncture, instead of inserting a swab containing oral fluid intoopening (20) in the cover an insert for receiving whole blood (FIGS.17A, 17B, and 18) is placed into the opening. This insert contains afunnel-like component in its top section (FIG. 17A). The top portion ofthis funnel is wide and permits the user to easily place the fingerstuck by the fingerstick lancet into the upper portion of the funnel sothat two drops of free-falling whole blood be collected into the funnelto contact the blood receiving membrane at location 65 (FIG. 18) heldbetween top and bottom portions of the insert (FIGS. 17A and 17B). Thespecial blood receiving membrane (e.g. LF1, GE-Whatman, Florham Park,N.J., USA) separates serum or plasma portions of the whole blood in theleading front of wicking down the membrane from cellular components ofwhole blood that migrate more slowly. The whole blood flows by capillaryaction from location 65 (FIG. 18) toward the other end of the membraneto saturate location 66 (FIG. 18) of the membrane with acellular serumor plasma. This method of collecting the whole blood into the testdevice helps to eliminate test failures due to improper transfer ofblood drops from the portion of the finger penetrated by the lancet tothe medical device.

The migration of the whole blood sample along the membrane to separatecellular from acellular components takes 4 minutes before an area ofmembrane saturated with serum or plasma from the sample overlies theo-ring of the device, that in combination with the dilution port whendepressed and locked in place, isolates an area of serum saturatedmembrane for testing. The o-ring in the midpiece for the whole bloodcollection device is of smaller dimensions (43, FIG. 16D) than theo-ring used for the oral fluid rapid test and combines with a dilutionport to isolate an area of saturated membrane that approximates half thesurface area of saturated membrane than is used in the oral fluid test.This partially adjusts for the approximately four to six-fold lowerconcentrations in gingival crevice fluid as compared to serum or plasma.The opening in the top of the dilution port that accommodates the vialor syringe containing running buffer is the same size in the dilutionport used for testing whole blood as in the dilution port for oralfluid. The base of the whole blood test device is identical to the baseof the oral fluid test device. The time to a readable test result foreach test is 10 minutes. For the oral fluid test, this time is taken upcompletely by migration following addition of diluent buffer. For thewhole blood test, four minutes is required for initial migration thatseparates acellular from acellular components, and the migrationfollowing addition of diluent running buffer requires an additional sixminutes to achieve a readable result. Both tests do not lose theirreadability for several days to weeks following test completion.

The following examples are intended to illustrate but not limit thescope of this invention.

EXAMPLES Example 1 Immunoreactivity of HIV Peptide-BSA Protein ConjugateAntigen

Peptide-BSA protein covalent conjugates containing peptides with thesequences found in the immunodominant portion of HIV-1, sequence region597-610 of gp41, as described in U.S. Pat. No. 5,260,189, (Formoso,Olsen, and Buchanan) and by Dorn et al (J. Clin Microbiology 38:2, pp.773-780, February 2000) were striped at approximately 1.5 ul/cm of a 250ug/ml concentration of total protein in 0.1N PBS, pH 8.0 containing0.05% sodium azide, onto nitrocellulose (Hi-Flow Plus HF-120, MilliporeCorporation, Bedford, Mass., USA) at approximately 1.5 ul/cm, and driedfor 10 minutes at 35 degrees C. under vacuum, followed by further dryingand storage at room temperature in the presence of anhydrous calciumsulfate desiccant (Drierite Company, Xenia, Ohio, USA). These stripednitrocellulose strips were then laminated, with overlapping to permitcapillary flow, to an absorbent pad (CF-4, GE-Whatman, Florham Park,N.J., USA) on the downstream end, and to a wicking glass membrane on theupstream end (Grade 142, Ahlstrom Filtration, LLC, Mount Holly Springs,Pa., USA) using a single-sided polyester-adhesive tape (ARcare 8160,Adhesives Research, Inc., Glen Rock, Pa., USA). Approximately 0.25 ODunits of particulate conjugate of Protein A bound to 40 nm colloidalgold (BA.PAG40, British Biocell International, Cardiff, UK) were mixedwith 1 ml of normal human serum diluted 1:100 with running bufferconsisting of 50 mM PO4 (Spectrum Chemical Manufacturing Corp., Gardena,Calif., USA), pH 7.4 with 0.1N NaCl (Spectrum), 0.05% Na azide(Spectrum), 0.1% Bovine Serum Albumin (IgG-free, protease-free, JacksonImmunoresearch Labs, West Grove, Pa., USA) and 2% Tween 20 (SigmaUltra,Sigma-Aldrich, Inc., St. Louis, Mo., USA). As described by Rosenstein etal. in U.S. Pat. No. 4,855,240, in addition to the BSA, a sugar such assucrose or trehalose was added to provide concentrations of 2% to 15% tominimize agglutinations of the particulate conjugate in the presence ofthe diluted serum and to decrease undesired interactions betweenparticulate conjugate and the laminated membranes of the flow path.

Diluted human serum containing antibodies to HIV produced a visiblereddish-purple line against a white background at the site of the HIVpeptide-BSA protein antigen immobilized on the nitrocellulose portion ofthe laminated test strips and no such line developed with similardiluted human serum that did not contain antibodies to HIV.

Immunoreactivity of the HIV peptide-BSA conjugate antigen was furtherevaluated using monoclonal antibodies. Monoclonal antibodies proven torecognize the immunodominant antigenic domain of HIV, specifically theamino-acid sequence regions 597-617 or 592-606 of gp41 of HIV-1,monoclonal antibodies T32 and F240 respectively, were each obtainedthrough the NIH AIDS Research and Reference Reagent Program, Division ofAIDS, NIAID, NIH: T32 Monoclonal Antibody (Cat. No. 11391) from Dr.Patricia Earl, NIAID; HIV-1 gp41 Monoclonal Antibody (F240) from Dr.Marshall Posner and Dr. Lisa Cavacini. The published references for eachof these monoclonal antibodies are T32—Earl et al., J. Virol. 71 (1997):2674-2684, and Cavacini et al., AIDS Res Hum Retroviruses 14:1271-1280,1998. The overlapping region of these two monoclonal antibodies issequence region 597-606. This region contains a sequence CSGKLIC that isrelatively preserved among widely variant geographic isolates of HIV,and this sequence is contained within the peptides described in U.S.Pat. No. 5,260,189, (Formoso, Olsen, and Buchanan). Monoclonal antibodyT32 recognizes a linear epitope within this region (see Earl et al., J.Virol. 71 (1997): 2674-2684) and monoclonal antibody F240 recognizes aconformational epitope (see Cavacini et al., AIDS Res Hum Retroviruses14:1271-1280, 1998), presumably formed by an S—S bond between the twocysteines in the peptide sequence CSGKLIC.

HIV-1 peptides containing the sequence CSGKLIC were conjugated to BSA asdescribed in U.S. Pat. No. 5,260,189 and this peptide-BSA conjugate wascustom bound to 40 nm colloidal gold by British Biocell International(BBI, Cardiff, UK). Monoclonal antibodies F240 and T32 were immobilizedon nitrocellulose HF 120 (Millipore Corp., Bedford, Mass., USA) bystriping at various concentrations monoclonal antibody in 0.1N PBS, pH7.4 with 0.05% Na azide, followed by drying at 35 degrees C. undervacuum for 15 min followed by room temperature desiccation in thepresence of anhydrous calcium sulfate desiccant (Drierite Company,Xenia, Ohio, USA). The custom bound HIV peptide-BSA conjugates werediluted to 2.5 OD concentration in 2 mM borate pH 7.1 with 0.0025% Naazide and 15% trehalose and coated onto S-14 conjugate pads (GE-Whatman,Florham Park, N.J., USA) that had been pre-blocked with 0.0025% Na azide(Spectrum Chemical, Gardena, Calif.), 0.1% bovine serum albumin(Probumin, Diagnostic Grade, Celliance, Serologicals Corporation,Kankakee, Ill., USA) and the coated S-14 conjugate pads were dried at 37degrees C. under vacuum for 30 min followed by room temperature dryingin the presence of anhydrous calcium sulfate desiccant (DrieriteCompany, Xenia, Ohio, USA). Evaluation strips were laminated usingARcare 8160 (Adhesives Research, Inc., Glen Rock, Pa., USA) withmonoclonal antibody-bound nitrocellulose connecting to an absorbent paddownstream, and upstream to the conjugate pads containing BBI-prepared40 nm colloidal gold HIV peptide-BSA conjugate antigen, which overlappedfurther upstream to a wick upstream (Grade 142, Ahlstrom Filtration,LLC, Mount Holly Springs, Pa., USA). These test strips were evaluated byapplying running buffer PBSAAT consisting of 50 mM PO4 (SpectrumChemical Manufacturing Corp., Gardena, Calif., USA), pH 7.4 with 0.1NNaCl (Spectrum), 0.05% Na azide (Spectrum), 0.1% bovine serum albumin(IgG-free, protease-free, Jackson Immunoresearch Labs, West Grove, Pa.,USA) and 2% Tween 20 (SigmaUltra, Sigma-Aldrich, Inc., St. Louis, Mo.,USA).

Monoclonal antibodies T32 and F240 were immobilized on nitrocellulose(HF 120) at various concentrations in 0.1 N PBS, pH 7.4 with 0.05% Naazide and dried as described above. Monoclonal antibody T32 produced afaint line of binding of the HIV peptide-BSA conjugate antigen bound to40 nM colloidal gold when striped at 400 ug/ml concentration. Acomparable line of binding of the same HIV Peptide-BSA conjugate antigenwas achieved with F240 monoclonal antibody striped at 100 ug/ml. Thedisadvantages of this type of a positive control for a rapid test are(a) the HIV peptide-BSA antigen is not identical to the same antigenused in the flow path to detect antibodies specific to HIV, since it hasbeen modified through the procedures of binding it to colloidal gold and(b) this type of positive control does not simultaneously evaluate thereactivity of the particulate markers used in the flow path to detectantibodies specific to HIV. These disadvantages may be overcome byinstead coating the same native HIV peptide-BSA conjugate antigen tonitrocellulose in both flow paths, providing the monoclonal antibodiescan be recognized by the particulate marker.

Monoclonal antibody T32, which recognizes a linear epitope, produced alightly visible line at the site of immobilized HIV peptide-BSA proteinantigen on the nitrocellulose laminated strips when 15 micrograms ofantibody was reacted with 0.20 O.D. units of 40 nm colloidal goldprotein G (BioAssay Works, LLC, Ijamsville, Md., USA) and migrated withrunning buffer PBSAAT (described above) downstream over the test strip.In contrast, the F240 monoclonal antibody produced a significantlystronger visible line of binding to the immobilized HIV peptide-BSAprotein antigen when only 10 picograms was reacted with the samecolloidal gold protein G and migrated over identically striped laminatednitrocellulose strips. This 1500-fold or greater potency of the F240monoclonal antibody suggests that the HIV peptide-BSA protein antigen isin cyclized form, and the reactivity of this antigen with humanantibodies is consistent with previous reports that the dominant epitopefor the human immune response to HIV infection is a conformationalepitope, presumably the S-S loop epitope contained within the HIVpeptide-BSA protein antigen used in this example.

Example 2 Evaluation of Stability of HIV Peptide-BSA Conjugate Antigenand Monoclonal or Polyclonal Antibody in Dried State for Use in RapidDiagnostic Tests to Prove Antigen Integrity in the Reagent PositiveControl Flow Path

Nitrocellulose striped with HIV peptide-BSA conjugate antigen as inExample 1 was stored at room temperature at 10-30% humidity levels andtested over time against both F240 monoclonal antibody and human serumcontaining antibodies to HIV. The antigen retained its reactivity formore than one year under these conditions.

2 ug of F240 monoclonal antibody was spotted onto a 0.7 cm×1 cm membranestrip of protein G 40 nm colloidal gold coated at 0.25 OD units/squarecm (BioAssay Works, LLC, Ijamsville, Md.), allowed to dry at roomtemperature and then stored at room temperature for 13 days withoutdesiccant. The contents of this membrane strip were then migrated overthe HIV peptide-BSA conjugate dried onto nitrocellulose, using runningbuffer as described in Example 1. The F240 monoclonal antibody andprotein G colloidal gold produced a strong line of immunoreactivity withthe HIV peptide-BSA conjugate antigen, confirming a degree of stabilityof the F240 monoclonal antibody when dried in the presence of thestabilizers present in the coated colloidal gold membrane strip.

F240 monoclonal antibody was then diluted to a concentration of 7.5ug/ml in a solution of 0.1N PBSAA (0.1 N PO4, pH 7.4 with 0.1 N NaCl,0.01% BSA (Jackson Immunoresearch Labs, protease and IgG free), 0.03% Naazide and 10% Trehalose (FlukaBiochemika, Sigma-Aldrich Corp., St.Louis, Mo., USA) and coated to three different membranes S14(GE-Whatman, Florham Park, N.J., USA), 8964 (Ahlstrom, LLC, Mount HollySprings, Pa., USA) and G041 (Millipore Corporation, Bedford, Mass., USA)and dried at 45 degrees C. with vacuum for 10 minutes, followed by roomtemperature drying and subsequent storage in the presence of anhydrouscalcium sulfate desiccant (Drierite Company, Xenia, Ohio, USA). Thesestabilized & dried F240 monoclonal was stable and showed no evidence ofdecreased potency at recognizing the peptide-BSA HIV antigen coated tonitrocellulose when repeatedly tested during two month period, and againone year later.

Polyclonal antibody was also evaluated for use in the positive controlpathway. Human CRP, goat anti-human CRP and rabbit anti-human CRP wereobtained from CalBioreagents Inc. (San Mateo, Calif., USA). The humanCRP was coated to nitrocellulose at 100 ug/ml in 20 mM Tris bufferedsaline pH 8.2 containing 0.005% BSA and 20 mM sodium azide. Test stripswere assembled to allow running buffer alone, normal human serum diluted1:100 in running buffer, or running buffer containing 55 ug/ml of goatanti-human CRP or 30 ug/ml rabbit anti-human CRP to run past mobilizableprotein G 40 nm gold (BioAssay Works) and then past the immobilizedhuman CRP. Strong lines of binding were observed between the particulatemarker and the solutions containing goat or rabbit anti-human CRP, butno binding occurred with normal diluted normal human serum or runningbuffer alone. This demonstrated that polyclonal antibodies can also beused to recognize known antigens downstream in rapid flow test positivecontrols. Conditions similar to those used for the F240 MAb would beexpected to permit these polyclonal antibodies to be dried in stabilizedmobilizable condition with adequate shelf life for incorporation in torapid lateral flow tests for use as a positive control.

Example 3 Development of a Method to Determine Whether an AdequateAmount of Immunoglobulin is Present in the Diluted Sample to AllowDetection of Specific Antibodies to an Antigen or Analyte of Interest,if Present

No currently available rapid tests for antibodies to specific antigensor other ligands contain controls to determine if an adequate amount ofimmunoglobulin is being evaluated to allow detection of specificantibody if present. This is an important omission because tests offingerstick or venipuncture whole blood may be falsely negative ifinsufficient blood is introduced into the test, and samples of oralgingival crevice fluid may lack sufficient immunoglobulin to allowaccurate detection of specific anti-HIV antibodies if the oral swabfluid sample is collected improperly.

Experiments were conducted using different immunoglobulin detectionreagents, one or more being bound to a particulate marker, and anotherimmobilized at the downstream end of the test window of the flow pathfor detection of specific antibodies, to determine whether a set ofimmunoglobulin binding agents could be identified that in correctconcentrations would demonstrate binding of particulate markers by themost downstream immobilized immunoglobulin detection reagent only ifimmunoglobulin concentrations in the diluted sample are sufficient toallow detection of antibodies to specific antigens, if present. Thecombinations evaluated are presented in Table 1.

Surprisingly, of the 13 combinations tested, only the combination ofboth Protein L and Protein G colloidal gold when reacted with samplescontaining or not containing immunoglobulin, was able to correctlydiscriminate the presence of immunoglobulin. This combination, whenreacted with monoclonal antibody F240 or with human sera diluted withrunning buffer, produced binding to rPAdownsteam, but did not bind tothe immobilized rPA downstream when simply diluted with running bufferin the absence of immunoglobulin (Table 1). The conditions for coatingthe recombinant protein A to nitrocellulose were 0.1N PBS, pH 7.4containing 0.01% BSA (Jackson ImmunoresearchIgG and protease free) and0.05% Na azide. Optimal concentrations for rPA coating under theconditions of these assays were 1 to 5 micrograms/ml. Under theseconditions, the rapid assay could correctly discriminate whether thetest sample contained sufficient quantities of immunoglobulin to belikely to demonstrate a line of binding of particulate marker toimmobilized antigen, if specific antibodies to that antigen are present,and to detect those samples that were inadequate in terms of containingsufficient immunoglobulin. This combination was used in subsequentexperiments in the devices described herein to determine adequacy of thetest samples.

TABLE 1 Combinations of Ig Binding Reagents to Evaluate Sample IgAdequacy Binding of Particulate Ig Binding Reagent Ig Binding ReagentsBound Ig Present (+) Marker to Immobilized Immobilized on Nitrocelluloseto Particulate Markers or Absent (−) IgG Binding Reagent Protein A -Sigma-Aldrich Protein A 40 nm Gold - BBI + + Protein A - Sigma-AldrichProtein A 40 nm Gold - BBI − + rProtein A - Repligen Protein A 40 nmGold - BBI + + rProtein A - Repligen Protein A 40 nm Gold - BBI − + Goatanti-human IgG - Equitech Protein A 40 nm Gold - BBI + + Goat anti-humanIgG - Equitech Protein A 40 nm Gold - BBI − + Goat anti-human IgG Fc -Equitech Protein A 40 nm Gold - BBI + + Goat anti-human IgG Fc -Equitech Protein A 40 nm Gold - BBI − + Goat anti-human F(ab′)2 -Jackson Protein A 40 nm Gold - BAW + + Goat anti-human F(ab′)2 - JacksonProtein A 40 nm Gold - BAW − + Goat anti-human F(ab′)2 - Jackson ProteinG 40 nm Gold - BAW + + Goat anti-human F(ab′)2 - Jackson Protein G 40 nmGold - BAW − + Chicken IgY anti-human IgG - Aves Protein A 40 nm Gold -BAW + + Chicken IgY anti-human IgG - Aves Protein A 40 nm Gold - BAW − +Chicken IgY anti-human IgG - Aves Protein G 40 nm Gold - BAW + + ChickenIgY anti-human IgG - Aves Protein G 40 nm Gold - BAW − + Chickenanti-human IgG - Gallus Protein A 40 nm Gold - BAW + + Chickenanti-human IgG - Gallus Protein A 40 nm Gold - BAW − + Chickenanti-human IgG - Gallus Protein G 40 nm Gold - BAW + + Chickenanti-human IgG - Gallus Protein G 40 nm Gold - BAW − + rProtein A -Repligen Protein G 40 nm Gold - BAW + + rProtein A - Repligen Protein G40 nm Gold - BAW − + rProtein A - Repligen Protein L 40 nm Gold -BAW + + rProtein A - Repligen Protein L 40 nm Gold - BAW − + rProteinA - Repligen Protein G + L 40 nm Gold - BAW + + rProtein A - RepligenProtein G + Protein L 40 nm Gold − − Evaluation of PotentialCombinations of Immunoglobulin Binding Reagents that might, in thecorrect concentrations, and only after the particulate marker boundImmunoglobulin Binding Reagent has reacted with Immunoglobulin andmigrated downstream to the reagent immobilized on nitrocellulose,demonstrate binding of the particulate marker bound reagent to thereagent immobilized on nitrocellulose. Reagent Sources are Sigma AldrichInc. (St. Louis, MO, USA), Repligen Corporation (Waltham, MA, USA),Equitech-Bio Inc.(Kerrville, TX, USA), Jackson ImmunoResearchLaboratories, Inc., (West Grove, PA, USA), Aves Labs, Inc. (Tigard, OR,USA), Gallus Immunotech, Inc. (Fergus, Ontario, Canada),

Example 4 Detection of Immunoglobulin in Human Gingival Crevice OralFluid Followed by Detection of Antibodies to HIV Peptide-BSA Antigen inHuman Gingival Crevice Oral Fluid from HIV Positive Volunteers

Human gingival crevice fluid was collected from an HIV negativevolunteer onto an LF1 membrane (GE-Whatman, Florham Park, N.J., USA) byrubbing the membrane along the top and bottom tooth-gum lines for 30seconds. This membrane was then placed onto midpiece (6, FIG. 1)containing size 010 o-ring (5, FIG. 1). This midpiece with membranesaturated with gingival crevice fluid was placed atop a test tube withthe bottom exit of the midpiece allowing passage of fluid into the testtube. A dilution port (2, FIG. 1) was then placed atop the membrane sothat when downward pressure was applied the compressive force betweenthe bottom surface of the dilution port (37, FIGS. 2B and 2C) and matingtop surfaces of the size 010 o-ring positioned beneath provided aperimeter of compression that isolated a region of membrane centripetalto the ring of compression that was saturated with gingival crevicefluid. Running buffer was then applied to opening 31 in the top ofdilution port (FIG. 9A) and through the central channel of dilution port(36, FIG. 3A) to pass through the isolated region of LF1 membrane toprovide a sample of gingival crevice fluid diluted approximately 1:69within the test tube. This fluid was then applied to a test striplaminated together to provide upstream to downstream orientation ofdiluted sample membrane (Ahlstrom 142, Ahlstrom LLC, Mount HollySprings, Pa., USA)—protein G 40 nm colloidal gold @ 0.25 OD units/cm² onpolyester membrane (BioAssay Works, Ijamsville, Md., USA)—nitrocellulose(HF120, Millipore Corporation, Bedford, Mass., USA) coated upstream withpeptide-BSA HIV antigen (Example 1 above) followed further downstream byrPA (Repligen)—absorptive pad CF5 (GE-Whatman, Florham Park, N.J., USA).The diluted gingival crevice fluid flowed down the strip and showed nobinding to the HIV antigen, but showed definite but weak binding to therPA, indicating detection of the presence of IgG in the sample. SinceIgA is a prominent immunoglobulin in human oral fluids, and sinceprotein L can bind IgA as well as IgG, the combination of both protein Land protein G 40 nm colloidal gold (both from BioAssay Works, LLC) werelaminated to the upstream portions of otherwise identical strips andtested with the diluted oral gingival crevice fluid. Again, no bindingof the particulate markers occurred to the HIV peptide-BSA antigen.However, further downstream the particulate marker complex produced asignificantly stronger line of binding to rPA than with protein Gcolloidal gold alone. This protein G-Protein L-40 nm colloidal goldcomplex had also been found to produce the strongest recognition linesfor bound Ig by Repligen recombinant Protein A, and the leastnon-specific binding between this rPA as compared to protein G-gold orprotein L gold alone (see Table 1 above), so this combination was usedthereafter for tests of immunoglobulin in oral fluid samples.

LF1 membranes (GE-Whatman) are fragile, and while useful for separatingcellular components from serum or plasma when testing whole bloodsamples from patients, they would likely fragment if used for collectingoral fluid samples. For this reason, a fabric was sought that is clean,takes up fluid very rapidly within its void spaces, is capable of beingultrasonically welded to hold the fabric around swabs of designs such asthat shown in FIG. 6A, and is capable of releasing the fluid withprotein components present when tested in the test device (FIG. 1).Vectra AlphaSorb 10, double-knit fabric, used as swabs in clean roomenvironments (ITW Texwipe, Mahwah, N.J., USA) was evaluated for thispurpose. The AlphaSorb material was cut into strips that could be tapedover a gloved finger, and the gloved finger with fabric strips attachedwas used to rub the tooth-gum line both top and bottom. The strips wereremoved from the taped finger, placed between dilution port and o-ringin midpiece over a test tube as described above, and diluted with aknown volume of running buffer (see Example 1 above) and the dilutedgingival crevice fluid was run over strips laminated with overlaps toallow capillary flow as described above from upstream to downstream asfollows: diluted sample membrane—to protein L 40 nm colloidal gold—toprotein G 40 nm colloidal gold—to HF 120 nitrocellulose containingimmobilized HIV peptide-protein antigen followed downstream by rPA(Repligen)—to absorptive pads of CF5 glass-cellulose (GE-Whatman). Theperformance of diluted gingival crevice fluid samples collected on theAlphaSorb fabric were compared to oral fluid swabs sold commercially byCalypteBiomedical Corporation (Portland, Oreg., USA). The recognition ofIg in the diluted samples by rPA was strong and equivalent for samplescollected from AlphaSorb fabric and the commercial Calypte swabs.Another diluted fluid sample, using the transport buffer provided byCalypte instead of the running buffer described in Example 1, also gavea line of recognition of Ig by rPA that was equal or slightly lessstrong than running buffer, but the diluted sample migrated more slowlythrough the membranes and provided a slightly yellow background ascompared to the white background resulting from use of running buffer.

It was also observed that if the AlphaSorb fabric was left to remaineither at refrigeration temperature or at room temperature for 5 hoursbefore diluting the samples contained therein with running buffer, mostof the immunoglobulin within the samples had adhered to the fabric andwas not recoverable for testing. In contrast, if the directions fromCalypte were followed, and the Calypte Messenger Transfer swabs wereimmediately immersed and rotated to remove sample into the vial oftransport fluid provided, the sample diluted with transport fluidcontained sufficient immunoglobulins to detect antibodies to HIV withinthe transport fluid diluted sample.

To test the ability of the AlphaSorb fabric to absorb and releaseantibodies reactive with the peptide-BSA HIV antigen, human volunteersthat were known to be HIV positive from laboratory tests were recruited.Three volunteers were tested using the method of AlphaSorb fabric stripstaped over a gloved finger to collect the gingival crevice oral fluid.After informed consent was obtained the positive volunteers werecompared to a known HIV negative volunteer during each test running. Thediluted samples from each volunteer, obtained by using running buffer toobtain and dilute an isolated area of fabric using the compressedperimeter method described above, were tested over strips consistingfrom upstream to downstream of diluted sample membrane-particulatemarker pad containing Protein L and Protein G 40 nm colloidalgold—nitrocellulose coated upstream with peptide-BSA HIV antigen, anddownstream with recombinant protein A—absorption pad. Volunteer 1 had aviral load of 5950 and a CD4 count of 481. Volunteer 2 had a viral loadof 693 and a CD4 count of 508. Volunteer 3 had a viral load of 1150 anda CD4 count of 557. All gingival crevice oral fluid samples from HIVpositive and HIV negative volunteers showed strong lines of Igrecognition by rPA, indicating adequate immunoglobulin in the samplestested. Each of the three HIV positive volunteers produced an easilyrecognizable line of binding of particulate marker to the peptide-BSAantigen in the test strips, whereas no line of particulate markerbinding was observed any of the three times the HIV negative volunteerprovided an oral gingival crevice fluid.

Example 5 Use of Rapid Test Device to Detect Human Antibodies to HIVPeptide-BSA Conjugate Antigen in Human Gingival Crevice Oral Fluid

The membranes pre-coated with test reagents as described above wereassembled into the oral fluid rapid test device as illustrated in thefigures. The swab illustrated in FIG. 6 that contained AlphaSorb fabricsewed into place around a plastic swab frame designed as shown in FIG. 6was used by an HIV positive volunteer to obtain an oral fluid sample fortesting. The portion of the swab containing the AlphaSorb fabric wasused to collect gingival oral fluid by swabbing for 20 seconds each boththe upper and lower tooth-gum lines. The swab containing the gingivalcrevice oral fluid was then placed immediately into the test devicethrough opening 20 (FIGS. 5B and 5C). The yoke (1, FIG. 1) was removedand the dilution port (2, FIG. 1) was depressed into the down and lockedposition (FIGS. 9D, 9E, 10C, and 11). This isolated an area of fabricsaturated with oral gingival crevice fluid to sample for the presence ofantibodies to HIV. An 800 microliter volume of running buffer was pushedthrough central channel 36 (FIGS. 3A and 10C) of the dilution port froma syringe fitted into opening 31 in its top surface (FIG. 9A). Thisfluid passed through the isolated membrane and out the undersurface ofthe midpiece onto the diluted sample membrane from which it passed overtwo separate pathways, the positive reagent control pathway and thepathway to detect antibodies specific for HIV-1 peptide-BSA antigen andfurther downstream to confirm that the sample contained adequateimmunoglobulin. The test device performed smoothly and the test resultwas readable in less than 10 minutes from the time of adding the runningbuffer diluent. In the pathway to detect specific antibodies and confirmsample adequacy a positive line appeared over both the immobilized HIVantigen and the rPA. In the positive control reagent pathway a strongline developed over the immobilized HIV antigen. Both the HIV antigenitself and the conditions of coating the antigen to nitrocellulose wereidentical in both pathways. The control results confirmed that thecollected sample was adequate, and that the critical reagents wereworking at the time of performing the test. These critical reagentsincluded the antigen, the particulate marker reagent, and the driedmobilizable monoclonal antibody used to recognize and bind to antigen inthe positive control pathway, regardless of whether or not antibodies toHIV antigen were present in the test sample. With these necessarycontrols all indicating a working test, it was possible to interpret theline of reaction to the HIV antigen in the specific antibody detectionpathway as a TRUE POSITIVE.

Example 6 Use of Rapid Test Device to Detect Human Antibodies to HIVPeptide-BSA Conjugate Antigen in Human Fingerstick or Venipuncture WholeBlood

A version of the device for testing whole blood was used to test an HIVpositive volunteer that had been under treatment for more than 20 years.He was relatively healthy and had a CD4 count of 600 and an HIV viralload of less than 2500. After lancing the volunteer's finger with apressure-activated lancet (Becton Dickinson, Franklin Lakes, N.J., USA),three drops of blood were transferred by bulb transfer pipet from thevolunteers fingertip to a sample well in the device. Beneath the samplewell was one end of a strip of LF1 membrane (GE-Whatman) of dimensions11 mm wide×39 mm long. Capillary flow of the whole blood over themembrane took approximately 5 minutes, with the acellular componentspreceding the trailing cellular components, to provide after 5 minutesat the downstream end of the LF1 membrane an area of membraneapproximately 12 mm×11 mm that overlay an o-ring size 008 sitting on topof the device midpiece. The yoke was removed, the dilution portdepressed and running buffer fluid was introduced into the dilution portopening under pressure which produced a diluted serum sample to thediluted sample membrane below the midpiece as described in Example 5.The diluted sample then flowed over the two paths described in Example 5as described above. After 5 additional minutes, the test was readable.There were strong easily discernable lines against a white background inboth control areas, indicating intact antigen and an adequate sample. Inaddition, there was an easily readable positive result line on theimmobilized HIV antigen in the specific antibody pathway, allowing aninterpretation of the results as a TRUE POSITIVE.

Example 7 Use of the Test Devices with Inadequate Samples, withMembranes Striped with Antigen that has Lost Immunoreactivity or withMembranes that Contain Particulate Marker Reagents that are Inactive, asCompared to Other Tests that are Fully Functional

In this example, the device test platform is used as described inExamples 2, 3, and 7. The reading window with rounded edges overlies anitrocellulose test and control strip that contains immobilized antigenupstream that is used to detect specific antibodies in the test, andcontains immobilized recombinant protein A downstream as a control todetect the presence of sufficient immunoglobulin in the diluted sample.The rectangular reading window overlies a nitrocellulose control stripof the reagent positive control pathway onto which the same antigen asused in the test strip has been immobilized under the same conditionsand in the same location as in the nitrocellulose strip beneath thereading window with rounded edges. Upstream along the reagent positivecontrol pathway Monoclonal Antibody F240 has been added to the bridgingmembrane (7, FIGS. 1 and 12) containing particulate marker of protein Gbound to colloidal gold as described in Example 2. No monoclonalantibody has been added to the particulate marker pad upstream in thespecific antibody detection pathway.

FIG. 14A—INVALID test due to failure of critical test reagents. Thisfigure illustrates the results obtained, as viewed through the devicecover, after tests are run in which the immobilized antigen hasdeteriorated, the particulate marker reagent has become inactive, ormonoclonal antibody dried onto membranes upstream in the reagent controlpathway has deteriorated. No line is present over the immobilized HIVantigen in either path window, and a single easily discernable line isseen through window 16 at the downstream end of the pathway to test forspecific antibody and sample adequacy.

Without a positive control path, this test result would have beenfalsely interpreted as NEGATIVE, not containing antibodies to HIV.However, with the positive control path, the user learns that somecritical reagent required for a proper test is not working. Thereforethe test must be read as INVALID and not interpretable. This saves theuser from falsely deciding that the test indicates that no antibodies toHIV, since this interpretation cannot be made without functioningcritical test reagents.

FIG. 14B—INVALID test due to use of an inadequate sample that containsinsufficient immunoglobulin. FIG. 14B illustrates a 2D schematic of theresults obtained as viewed through the cover of the oral fluid rapidtest device after it has been used to test a sample that contained nooral fluid. A dry swab was placed into the test device and the fluid runthrough the test was only running buffer. A strong line is present overthe immobilized HIV antigen in the results window 17 for the positivecontrol reagent path, but no lines are observed to demonstrate bindingof particulate marker to either immobilized antigen or to theimmobilized rPA Ig binding reagent in window 16 over the results area ofthe specific antibody and sample adequacy path.

Without a control to evaluate the presence of an adequate amount ofimmunoglobulin in the sample being tested for antibody to HIV, this testresult would have been falsely interpreted as NEGATIVE, not containingantibodies to HIV, and the user would conclude that individual whoprovided the sample was negative for antibodies to HIV. However, withthe control to evaluate sample adequacy, the user learns that the sampletested was inadequate to allow a result interpretation. Therefore thetest must be read as INVALID and not interpretable. This saves the userfrom possibly mistakenly concluding that the person that presented thedry swab for testing had no antibodies to HIV.

FIG. 14 C—VALID true positive test. FIG. 14C illustrates a 2D schematicof the results obtained, as viewed through the cover of the oral fluidrapid test device after it has been used to test an oral fluid samplethat contained antibodies to HIV. A strong line is present over theimmobilized HIV antigen in the results window 17 for the positivecontrol reagent path. Also, clear lines of particulate marker bindingare seen to both immobilized antigen, and to immobilized rPA Ig bindingreagent, through window 16 of the specific antibody and sample adequacypath.

This line of particulate marker binding to immobilized antigen in window16 of the sample adequacy and specific antibody pathway may beinterpreted as a TRUE POSITIVE since the control lines indicate that allcritical reagents are working, and that the sample tested containedsufficient immunoglobulin for evaluation.

FIG. 14 D—VALID true negative test. FIG. 14D illustrates a 2D schematicof the results obtained, as viewed through the cover of the oral fluidrapid test device after it has been used to test an oral fluid samplethat contained no antibodies to HIV. A strong line is present over theimmobilized HIV antigen in the results window 17 for the positivecontrol reagent path. Also, a clear lines of particulate marker bindingis seen to immobilized rPAIg binding reagent, through window 16 of thespecific antibody and sample adequacy path. However, there is no line ofparticulate marker binding to immobilized antigen as viewed throughwindow 16.

This lack of particulate marker binding to immobilized antigen in window16 of the sample adequacy and specific antibody pathway may beinterpreted as a TRUE NEGATIVE since the control lines indicate that allcritical reagents are working, and that the sample tested containedsufficient immunoglobulin for evaluation.

Example 8 Evaluation of Whether Monoclonal Antibody to HIV-1 Peptide BSAAntigen is Sufficient to Produce a Discernable Line of ParticulateMarker Binding with Recombinant Protein A, when Used in Concentrationsof Example 3

Recombinant Protein A (rPA) was immobilized on nitrocellulose strips at3.5 micrograms per ml downstream from the HIV-1 peptide BSA antigenunder the conditions of Example 3. Monoclonal antibody F240 andcolloidal gold particulate marker were run past the antigen and rPAunder the conditions of Example 1. A distinct line of colloidal goldparticulate marker bound to the immobilized HIV-1 peptide-BSA antigen,but no reactivity was observed where the rPA was immobilized. Thisindicated that the degree of binding of rPA to the small amount ofmonoclonal antibody was insufficient to produce a discernable line ofbinding of colloidal gold particulate marker complexed with Protein L+G.

An identically coated nitrocellulose strip was reacted with the samecolloidal gold Protein L+G particulate marker complex under identicalconditions to example 3 but an HIV-1 positive serum sample wassubstituted for the monoclonal antibody. A distinct line of colloidalgold particulate marker binding was observed over both at theimmobilized HIV peptide-BSA conjugate antigen and over the immobilizedrPA. This indicated recognition of human immunoglobulin in the HIVpositive serum by rPA, and that the concentration of rPA used boundsufficient immunoglobulin in the test serum to produce a line of bindingof colloidal gold marker complexed with Protein G and Protein L.

Together, these results indicate that controls for the rapid test forboth sample adequacy (sufficient immunoglobulin) and reagent reactivitymay be run on their own strip that is separate from the strip used todetect specific antibodies to analytes. This control strip may containboth immobilized rPA to indicate that the tested sample containsadequate amounts of immunoglobulin, and one or more binding pairsforanalytes, each consisting of a mobilizable monoclonal antibody andits specific immobilized analyte. Providing the monoclonal antibodiesused are of sufficient potency that the amounts used do not result in adiscernable line of colloidal gold at the immobilized rPA position, therPA may be used on the same control strip as the reagent reactivitypathway. Otherwise, sample adequacy for immunoglobulin content can beevaluated by placing the rPA on a pathway that does not receivemonoclonal antibodies.

Example 9 Devices that Prevent Backflow from Absorbent Pad to UpstreamAreas of Flow Path, Thereby Lengthening the Time Window During whichResults May be Reliably Observed

The length of time over which the results of a rapid test may be readaffect the usefulness of a test in different settings. For example, in abusy emergency room or in a hectic outpatient clinic it may not bepractical to read a test result exactly 10 minutes after beginning thetest and no later than 12 minutes! Current FDA approved rapid tests forHIV have reading windows of only two minutes (Trinity Unigold—between 10and 12 minutes) five minutes (Inverness Determine—between 15 and 20minutes) or twenty minutes (OraSureOraquick Advance—between 20 and 40minutes).

For designs that use an absorbent pad incapable of retaining all of theliquid introduced into the rapid test, backflow may occur that obscuresreliable reading of the test results. For this reason, in oneembodiment, the rapid test device includes a large absorbent pad. Thisembodiment is shown in FIG. 16A. Well 67 measures 20.5 mm wide×39 mmlong×2.45 mm deep. An absorbent pad of this dimension composed ofAhlstrom grade 237 cellulose is capable of retaining approximately 1.1ml of fluid. FIG. 16C illustrates the same design with test membranes inplace in which flow during performance of the rapid test is fromupstream areas on the right side of the device to downstream areas onthe left side of the test device. All fluid introduced upstream into thetest is effectively retained in the downstream absorbent pad with thisdesign and no backflow occurs, and test results are reliably observedwithout backflow interference for weeks rather than minutes.

Example 10 Device Designs to Prolong Delivery of Particulate Marker,with Resulting in Increased Sensitivity of Rapid Lateral Flow Testswhile Still Maintaining the Simplicity of Few Steps for Ease of Use

Conventional lateral flow assays for antibodies currently availableconsist of single test strips containing all components positionedin-line and in fluid communication by capillary action from upstream todownstream consisting of: sample receiving pad, mobilizable marker thatdetects immunoglobulin in the test sample, immobilized antigen that willbind specific antibodies being detected, and absorbent pad to take upthe liquid sample added upstream and maintain the flow direction of thetest from upstream to downstream (see Rosenstein, U.S. Pat. No.5,591,645). With this design, the first portion of the liquid sample tointeract with the mobilizable marker consists of a high concentration ofmobilizable marker relative to the amount of antibody in the liquidsample. As the test proceeds, the mobilizable marker binds to antibodyin the liquid sample and the marker-antibody complexes migrate down thestrip past the immobilized antigen. Subsequent amounts of liquid samplefind little remaining mobilizable marker with which to interact, andthough antibodies in this portion of the sample may bind to immobilizedantigen downstream they are not visually recognized since allmobilizable marker has already migrated downstream from the immobilizedantigen.

Rosenstein and Bloomster first described the advantages of firstapplying sample to immobilized analyte in lateral flow assays prior topassing tracer over the same immobilized analyte (U.S. Pat. No.4,855,240). These advantages were further expounded by Bernstein et al.(U.S. Pat. No. 5,824,268) and subsequently by Esfandiari (U.S. Pat. No.7,387,890 B2). Esfandiari (Dual Path Platform Lateral Flow) and verticalflow assays that provide interaction of immobilized antigen with liquidsample containing antibodies prior to interaction with visual markerhave claimed greater sensitivity (see Chan, U.S. Pat. No. 7,531,362 B2).

Experiments were conducted to determine whether design modifications tothe test device might allow more antibody (analytes) in the liquidsample to bind to downstream immobilized antigen (antibody bindingligand) prior to interaction with mobilizable marker.

To determine the probable distribution of antibodies within the dilutedsample produced by the test device of FIG. 1 and the method of U.S. Pat.No. 7,364,914 B2 (Buchanan), a concentrated solution of red foodcoloring (McCormick & Co. Inc., Hunt Valley, Md.) with a peak wavelengthabsorption at 535 nm, was used to saturate 2 layers of Alpha Lite Fabric(ITWTexwipe, Inc., TX 1008B) placed over the midpiece (6) with o-ring(5) of the test device (FIG. 1) containing a diluted sample membrane (8)but no test or control strips. The dilution port (2, FIGS. 1 and 10) waspushed down into locked position to compress a perimeter of the isolatedarea of saturated alpha lite fabric (FIGS. 10B and 10C) and 1 ml ofrunning buffer (sample diluent, Example 1) was passed through theisolated area of alpha lite fabric saturated with a concentratedsolution of red food coloring which removed it from the alpha lite andsimultaneously passed the diluted solution down into the diluted samplemembrane lying beneath the midpiece. The device was opened and thediluted sample membrane was visually inspected. Dividing the dilutedsample membrane into quarters, the peripheral 25% ends of the dilutedsample membrane were dark red, and the inner 25% portions outside of acentral white circular area corresponding to the exit flow path from themidpiece were a lighter red. These observations indicate that thehighest concentration of diluted sample migrate from the center of thediluted sample membrane to the outside edges, and subsequent runningbuffer contacts less sample within the alpha lite fabric and these lowerdilutions distribute closer to the exit point from the midpiece.

The diluted sample membrane (DSM) was cut into fourths consisting of twoouter and two inner portions. Each was placed into a test tube and 1 mlof DI water was added, and each test tube was then mixed for 20 secondson a vibratory shaker. Aliquots of each tube were then read in aspectrophotometer for absorbance at 535 nm wavelength. The two outer 25%portions produced readings of 1.201 and 1.303 for an average absorbanceof 1.252. The two inner 25% portions produced readings of 0.665 and0.785 for an average absorbance of 0.725 confirming the visualobservation that higher concentrations of diluted sample were found inthe peripheral portions of the DSM membrane as compared to portions nearto the exit well from the midpiece.

To estimate the approximate differences in concentration of dilutedsample between peripheral and inner portions of diluted sample membranewhen liquid samples are diluted according to U.S. Pat. No. 7,364,914 B2(Buchanan), a standard curve of dilutions on concentrated red foodcoloring was performed, beginning with 535 nm absorbance of 1.255 anddiluting therefrom. The standard curve indicated that the averageconcentration of diluted sample in the inner two 25% portions of the DSMwas approximately 50% of the average concentration of the red foodcoloring in the outer two 25% portions of the DSM. Thus, duringperformance of the rapid test using the method and rapid test devicedesign described herein, concentrations of diluted sample to migrateover the two pathways, the pathway to evaluate reagent reactivity andthe separate pathway to detect antibodies (analytes) to specificantigens (antibody binding ligands) are at their highest levelinitially, and then the concentrations of diluted sample decrease toapproximately half of the initial levels on average during delivery ofthe second half of the diluted sample through the reagent reactivity andspecific analyte detection pathways.

FIG. 16A illustrates a design modification for the rapid test device toallow a portion of the particulate marker to be delivered throughoutmost of the period of fluid flow from diluted sample membrane toabsorbent pad. In FIG. 16A, the more central of the two dilution portreceptacles (29, FIG. 8B) in the base has been moved centrally, whichallows room for a long strip containing mobilizable colloidal goldprotein G+L particulate marker (55, FIG. 16B) to overlap partially alongits upstream long edge with the diluted sample membrane and to restpredominantly upon a plastic surface of the base (49, FIG. 16A) adjacentthe well for the diluted sample membrane. Only its ends are directly inline with upstream portions of the test and control flow paths (FIG.16C). The portion of particulate marker strip that is directly in linewith the separate reagent reactivity and specific analyte detection flowpaths interacts with the initial higher concentrations of diluted sampleand flows downstream in line with the leading edge of fluid flowingthrough the test, as is typical for conventional lateral flow diagnostictests. However, for the particulate marker in the more central regionsof the long particulate marker strip to reach the reagent reactivity andseparate specific analyte detection flow paths, it must interact withdiluted fluid from the central regions of the diluted sample membrane.These more central regions contain lower concentrations of dilutedsample (FIG. 16F).

As shown in FIGS. 16E and 16F, the methods and devices of this inventionallow particulate marker from pad 67 to interact with diluted samplemembrane (56) over a variety of diluted sample concentrations rangingfrom 0% to 100% of the most concentrated dilution produced by the testdevice. This is in contrast to conventional particulate marker pads,such as pad 68, FIGS. 16E and 16F, that are in-line and the same widthas the downstream membranes of the pathway, and interact only with theleading edge of a sample dilution, which is generally of higherconcentration than subsequent sample to migrate over the test device. InFIG. 16F, conventional particulate marker pad 68 interacts only withsample dilutions ranging from 70-100% of full strength dilution. Incontrast, particulate marker pad 67 presents the same amount ofparticulate marker to sample dilutions of 0-10% of full strength at thecenter of the diluted sample pad, as it does to sample dilutions of10-40% one step from the center, and to sample dilutions of 40-70% nextfurther from the center, and to 70-100% of full strength sampledilutions near the point where it joins with the specific antibodypathway 53 (FIG. 16F). This provides more opportunity than withconventional lateral flow immunoassays to achieve optimal complexes ofmarker to immunoglobulin molecules such that the complexes contain fullyfunctional immunoglobulin molecules to recognize and bind to downstreamimmobilized antigen (analyte binding ligands) yet also form large enoughclumps of bound marker to provide optimal detection of specificantibodies (analytes) bound to immobilized antigen.

Another advantage of the use of a membrane for collecting diluted sample(FIGS. 16B, 16C, 16E, and 16F) instead of a well as used in U.S. Pat.No. 7,364,914 is that there is less likelihood of spillage of dilutedsample if the test device is bumped or used in a non-level surface. Themembrane holds the diluted sample within its void spaces, available forflow by capillarity into downstream membranes, but this ability to holdthe diluted sample on varying concentrations is not overcome by bumpingthe test while it is running or performing the rapid test on a less thanlevel surface.

When this design was compared to the more conventional use ofparticulate marker in lateral flow assays, particulate marker with themodified design was delivered to the test and control strips in moreintense amounts approximately 50% longer than with conventional lateralflow designs, and lower amounts of particulate marker continued to bedelivered to the test and control strips for approximately three timeslonger. When the test was disassembled after completion, the particulatemarker strip was consistently noted to be white, indicating nearcomplete removal from all particulate marker from the longer strip andits delivery to the specific analyte detection flow path (FIGS. 16E and16F) or to both the reagent reactivity and specific analyte detectionflow paths (FIG. 16A) membranes. This longer delivery period ofparticulate marker provides an opportunity for more antibodies withinthe diluted test sample to reach and bind to immobilized antigensdownstream prior to the last available particulate marker that mightrecognize this binding exiting the flow paths into the absorbent pad.

In another experiment, the time period during which colloidal goldparticulate marker was delivered to the end of the viewing window forthis modified design was 3 times longer than with the conventionallateral flow immunoassay design (Table 5, Example 12). In conventionallateral flow immunoassays all of the mobilizable particulate marker iscontained on an in-line strip between sample pad upstream andnitrocellulose membrane downstream and all of the particulate markerfrom the strip enters the lateral flow immunoassay with the initialdiluted sample flowing from the sample pad. After this initialinteraction between sample and particulate marker, sample continues toflow over the lateral flow immunoassay but there is little particulatemarker, if any, with which to interact.

With this modified design, diluted sample and particulate marker migratelaterally together into the downstream specific antibody (analyte)detection zones containing immobilized antigen (analyte binding ligands)where reactivity is observed through the test device windows. This is incontrast to the methods and devices of Rosenstein (U.S. Pat. No.4,855,240), Bernstein (U.S. Pat. No. 5,824,268) and Esfandiari (U.S.Pat. No. 7,189,522 B2—Dual Path Platform) where sample collection andpreparation and delivery to the test device are via a different pathwaythan the pathway used for particulate marker and the two pathways meetat the detection zone of immobilized antigen, with sample preferablyreaching the detection zone before marker. With this invention, there isno need to use separate application points for sample and buffer, thusretaining the simplicity and ease of use desired for point-of-care andat-home testing sites.

Example 11 Evaluation of Relative Sensitivity of Rapid Lateral FlowTests that Employ Prolonged Delivery of Particulate Marker During theReaction as Compared to Conventional Rapid Lateral Flow Tests

The rapid test device using the base of FIG. 16A and cover of FIG. 15Aand whole blood insert of FIG. 17 was used to detect antibodies to HIV-1peptide-BSA antigen in dilutions of monoclonal antibody F240 thatrecognizes the immobilized HIV-1 peptide-BSA antigen as described inExamples 1 and 2. Dilutions of the F240 monoclonal antibody in testrunning buffer (Example 1) such that 40 microliters of running bufferthat produced saturation of an LF1 membrane (Example 6) overlying theo-ring of the test device (FIG. 16D) contained 7500 pg, 750 pg, 75 pg or7.5 pg of monoclonal antibody. Once the LF1 membrane received thedesignated quantity of monoclonal antibody F240, the dilution port ofthe test device was pushed down into the locked position to isolate acircle of saturated membrane centripetal to the o-ring and 1 ml ofrunning buffer was delivered with pressure through the opening in thetop of the dilution port (2, FIG. 1) to remove and simultaneously dilutethe monoclonal antibody and deliver it to the diluted sample membranelocated adjacent the exit port of the midpiece on the undersurface ofthe midpiece. This action produced diluted F240 antibody dilutions thataveraged 1:200-1:250 dilutions of the concentration of F240 delivered tothe LF1 membrane, but contained higher concentrations of F240 near theends of the diluted sample membrane and lower concentrations than 1:250in the more central regions of the diluted sample membrane. Thesedilutions were then run over the two pathways of the FIG. 16C device,the reagent reactivity pathway, and the specific analyte (antibody)detection pathway. In one case the particulate marker was provided witha strictly in line upstream particulate marker pad as is typical forconventional lateral flow immunoassays (membrane 68, FIG. 16C). Inanother embodiment, the device of the invention provides a longer andthinner pad of particulate marker positioned as illustrated by pad67(FIG. 16 C). Observable line intensity over the immobilized HIV-1antigen was evaluated at 10, 15, and 20 minutes for each test design andthe results are summarized in Table 2.

TABLE 2 Sensitivity of new and conventional lateral flow immunoassayMethods and device designs for detection of monoclonal antibody to hiv-1New Lateral Flow Design Conventional Lateral Flow Design Line Intensity10 min 15 min 20 min Line Intensity 10 min 15 min 20 min Picograms F240Picograms F240 7500 5 5 5 7500 4 4 4 750 4 4 4 750 2.0 3 3 75 2.5 3 3 750 0.5 1 7.5 0 0.5 0.75

These results suggest a five to ten-fold improvement in sensitivity withthe New Generation Modified Design of this invention as compared toconventional lateral flow techniques for this embodiment.

Sensitivity differences between conventional lateral flow test devicedesigns and the new generation design of FIG. 16C were evaluated usingseroconversion panel PRB 945, obtained from SeraCare Life SciencesCorporation, Milford, Mass., USA. This panel contained 6 memberscollected at 0, 3, 7, 13, 15, and 20 days from the date of the firstbleed. HIV RNA by Roche PCR was detectable in all 6 members beginningwith 300 copies/ml in sample 1, 700/ml in sample 2, 9000 in sample 3 andgreater than 80,000 in samples 4-6. No antibodies were detectable by anytests, including 2010 instrument based tests, in samples 1-3, and noneof the six samples gave uniformly positive Western Blot tests. Sample 6gave a positive Western Blot result in one of three confirmatory WesternBlot tests, and the other two were indeterminate. Detection ofantibodies to HIV in sample 4 was possible with six of six 2010instrument based tests, with five of twelve 1997 instrument based tests,and with no rapid non-instrument based tests. Sample 5, taken 2 daysafter sample 4, contained antibody to HIV detectable by all six 2010instrument based HIV antibody tests, nine of twelve 1997instrument-based tests, and a single vertical flow-through rapidnon-instrument based 1997 test. Sample 6, taken 5 days after sample 5and 20 days after sample 1, contained antibodies to HIV-1 in all 2010instrument based tests, in 10 of 12 1997 instrument based tests, and ina single rapid vertical flow through rapid test.

Sample 6 was tested in the rapid test device of this invention (FIGS.15-18) with particulate marker delivered both in conventional lateralflow format, or in the new generation modified format designed todeliver particulate marker over a longer period during test development.The results are shown in Table 3.

TABLE 3 Sensitivity for Detection of Seroconversion by Conventional asCompared to New Device Designs and Methods incorporating New Size andOrientation of Particulate Marker Pads and Multiple Ratios and ExtendedDuration of Marker to Immunoglobulin Interactions New Lateral FlowDesign Conventional Lateral Flow Design Line Intensity 10 min 15 min 20min Line Intensity 10 min 15 min 20 min Sample 6 of SeraCare CorporationPRB 945 SeroConversion Panel tested by 1 1.5 2 0 0 0 Sample 5 ofSeraCare Corporation PRB 945 SeroConversion Panel tested by 0 0.5 1.0 00 0

These results indicate that sensitivity is increased using the newgeneration lateral flow design of this invention that deliversparticulate marker over longer periods of antigen antibody interactionsand allows test development with a greater range of immunoglobulinconcentrations relative to particulate marker than occurs with testsemploying conventional designs for rapid lateral flow immunoassays.

Example 12 Studies of the Effect of Sample Immunoglobulin Concentrationon the Intensity of Observable Binding of Particulate Marker toImmobilized Antigen and Recombinant Protein A, and Comparisons ofParticulate Marker Delivery Times with Conventional Rapid Lateral FlowDesigns as Compared to New Generation Lateral Flow Designs of thisInvention

The test device particulate marker design of FIG. 16C was used toevaluate reactivity of F240 with HIV-1 BSA-peptide antigen as influencedby the concentration of normal human serum in the test sample dilutedwith running buffer. The monoclonal antibody was coated in mobilizableform onto Millipore GFCP203000 (Bedford, Mass., USA) under theconditions of Example 2, and was mobilized by the sample migrating fromthe diluted sample membrane (56, FIG. 16C) downstream past mobilizedantigen, then recombinant protein A, and into the absorbent pad (50,FIG. 16C). The intensity of binding of particulate marker tonitrocellulose immobilized HIV-1 peptide-BSA antigen, and downstream torecombinant protein A, is shown in Table 4.

TABLE 4 Effect of Serum Concentration Tested on Intensity of ReactionObserved Observed Intensity of Serum Concentration Particulate MarkerBinding to of Diluted Sample Immobilized Antigen Recombinant Protein A1:25 Diluted Serum 0.75 0.75 1:80 Diluted Serum 2 2 1:200 Diluted Serum3 4

These results indicate that the amount of particulate marker availablein rapid lateral flow immunoassays is insufficient to bind all of theimmunoglobulin in serum specimens more concentrated than 1:80, and thatthese immunoassays is more likely to produce reliable results at serumconcentrations more dilute than 1:150.

In the rapid lateral flow immunoassays of this invention, the designshown in FIG. 16C may be expected to detect specific antibodies withhigher sensitivity than conventional lateral flow immunoassays forseveral reasons. One of these reasons is that particulate marker isdelivered over longer time periods of the assay with the new generationdesign than with conventional configurations, as that shown in FIG. 16E[conventional design pathway 52 (the reagent reactivity pathway)] withparticulate marker pad 68, versus new generation design pathway 53 (thespecific analyte detection pathway) with particulate marker pad 67)].This longer delivery of particulate marker allows recognition of analyte(antibody) in the diluted sample not previously bound to particulatemarker, or only sparsely bound, that has migrated to and bound toimmobilized analyte binding ligands (antigen) downstream and is thenrecognized by particulate marker when it later migrates by as it isreleased later by the new generation design (pad 67 and pathway 53, FIG.16C). Another potential reason for increased sensitivity is that analyte(immunoglobulin) concentrations are lower in the eluent from the dilutedsample membrane onto each downstream pathway during the latter half offluid flow from diluted sample membrane to absorbent pad, as furtherdescribed in Example 10 above. These lower concentrations ofimmunoglobulin may interact more favorably with particulate markereluting from the new generation pad designs (pad 67, FIG. 16C), duringthe second half of diluted sample migration during the test than withthe higher immunoglobulin concentrations present in the diluted thatmigrate past the particulate marker pad during the first half of samplemigration.

To directly compare the duration of particulate marker delivery todownstream test components from conventional and new generationparticulate marker pads a test with the particulate marker configurationshown in FIG. 16E was performed. In FIG. 16E, particulate marker pad 68is of conventional configuration and delivers its colloidal goldparticulate marker to pathway 52 (the reagent reactivity flow pathway).For comparison, particulate marker 67 is of new generation configurationand delivers is colloidal gold particulate marker to pathway 53 (thespecific analyte detection flow pathway). The flow patterns of dilutedsample delivered to area 57 of the diluted sample membrane, andspreading from there to saturate the membrane, are shown by four dashedlines 58 leading to pathway 52, and by six dashed lines 58 leading topathway 53 (FIG. 16E). In both pathways, initial flow from dilutedsample membrane into colloidal gold particulate marker pads is fromdiluted sample in the saturated ends of the membrane through theparticulate marker pads at the upstream end of each pathway through thebridging membranes (59, dashed lines) into the nitrocellulose strip(membrane containing immobilized analyte binding ligands) of eachpathway, and then down each flow pathway into the absorbent pad (50,FIG. 16E). However, later portions of the diluted fluid sample do notcarry any particulate marker into pathway 52 since all of the marker wasremoved with the initial diluted sample that migrated through pad 68. Incontrast, after all particulate marker has been removed from the portionof particulate marker pad 67 that is adjacent the upstream end ofpathway 53, there is still opportunity for continued flow of particulatemarker from the portions of pad 67 that are not immediately adjacent tothe upstream end of pathway 53, as shown by dashed lines 58 of FIG. 16E.Diluted sample migrates into particulate marker pad 67 from its overlapalong one edge with the diluted sample membrane, and continues migrationdown pad 67 until marker can enter the upstream portions of pathway 53via the bridging membrane 59 (dashed lines) connecting pad 67 to theupstream end of the nitrocellulose membrane (immobilized analyte bindingligand membrane) portion of pathway 53 (the specific analyte detectionflow pathway). When the duration of flow of visible particulate markercolloidal gold-protein G+L was observed for pathways 52 and 53 with thetest design of FIG. 16E, the results shown in Table 5 were obtained.

TABLE 5 Time periods observed for colloidal gold marker to reach andleave reading windows of rapid lateral flow assay with conventional ascompared to new design and orientation of particulate marker padsConventional New Time period for leading edge of visually 40 seconds 40seconds observable colloidal gold visual marker to reach downstream endof viewing window of test device Time period for last of visiblecolloidal  3 min 30 sec. 10 min 30 sec. gold marker to reach downstreamend of viewing window

The results of Table 5 demonstrate that a particulate marker pad of thenew design of this invention, such as pad 67 of FIG. 16E, can delivercolloidal gold marker to rapid lateral flow immunoassays for a timeperiod approximately 3 times longer than the time period during whichthis marker can be delivered from pads of conventional lateral flowconfiguration, such as pad 68 of FIG. 16E. This prolonged availabilityof particulate marker capable of recognizing downstream analytes(antibodies) bound specifically to immobilized analyte binding ligands(antigens) throughout the rapid test performance, allows recognition ofanalytes (antibodies) specific to immobilized analyte binding ligands(antigens) that may have bound to those analyte binding ligands(antigens) during minutes 4 through 9 of the test performance, which isnot possible with the conventional lateral flow designs for particulatemarker delivery to test strips. This increased recognition of antibodiesbound during 4-9 minutes of the test, in this example, may explain, atleast in part, the observed higher sensitivity of the new generationparticulate marker pad delivery method of this invention observed inTables 2 and 3 of Example 11.

Example 13 Use of a Specifically Designed Whole Blood Insert for TestingWhole Blood Samples within the Rapid Test Device

LF1 membrane (GE-Whatman, Florham Park, N.J.) was cut precisely to theinner dimensions over the platform and its open space of the whole bloodinsert (FIG. 17B, 62 and 64, respectively) and the top portion of thisinsert (FIG. 17A) was fitted to it to hold the membrane in place. Thewhole blood insert containing the L1 membrane was placed into positionin the rapid test device with the device design cover of FIG. 18 and thedevice design base of FIG. 16A, that contained a midpiece (6, FIG. 1)that contained size 008 o-ring within its top surface (23, FIG. 7). Acontact-activated lancet from Becton Dickinson (Becton Dickinson,Franklin Lakes, N.J., USA) was used to lance a volunteers fingertip andtwo free-falling drops of whole blood from the fingertip were allowed todrop onto the area of the LF1 membrane located beneath the opening inthe top piece of the whole blood insert (65, FIG. 18). After 4 minutes,this whole blood had migrated to the opposite end of the LF1 membraneand the area defined by the 008 membrane (66, FIG. 18) was enriched foracellular as compared to cellular components of the whole blood. At thistime, the yoke (1, FIG. 1) was removed from the dilution port (2,FIG. 1) and pressed down into the base receptacles (29, FIG. 8B) whereits hook arms (32, FIG. 9C) locked into position within the basereceptacle windows (30, 8C) of the dilution port hook arms (32S and 33S,FIG. 9C). In this locked position the bottom surface of the centralchannel opening of the dilution port (37, FIGS. 2B and 2C) appliescompressive force on the top surface of the membrane that overlies theo-ring within the top surface of the midpiece (5, FIGS. 7B and 7C). Thisforce is met by a corresponding opposing force from the o-ring on theundersurface of the membrane and together these forces compress andcollapse the LF1 membrane void spaces that are saturated with theacellular-enriched sample of whole blood, thereby isolating an area ofsaturated membrane centripetal to the ring of compression, in accordancewith U.S. Pat. No. 7,364,914. Thereafter, one ml of running buffer forthe rapid test was expressed under pressure via a vial or syringed thatformed friction leak-proof fit between the neck of the vial or exit ofthe syringe and the opening in the top surface of the dilution port(FIG. 2A). This running buffer traveled through channel 36 (FIG. 3A) toexit the dilution port overlying the isolated membrane, and continuedthrough the membrane removing the acellular-enriched blood sample andsimultaneously diluting it and delivering it through channel 25 of themidpiece (FIG. 7A) and exiting at 27 in the undersurface of the midpiece(FIG. 7C) onto the diluted sample membrane 56 at area 57 (FIG. 16B) andfrom there flowed over the pathways of the rapid test as illustrated byexample in FIGS. 13, 16C, and 16E.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. A method for detecting an analyte in a liquid sample and checking thereactivity of the method reagents comprising: (a) isolating a portion ofa compressible membrane containing at least a portion of a liquid sampleto be analyzed by applying compressive force on top and bottom surfacesof the membrane along a perimeter of an area to be isolated therebydefining a non-compressed area of the membrane centripetal to thecompressed perimeter; (b) delivering a pre-determined amount of liquiddiluent under pressure to the isolated portion of the membrane torelease at least a portion of the liquid sample from the isolatedportion of the membrane to provide a diluted sample; (c) contacting thediluted sample with a second porous membrane having a first and secondend, wherein the diluted sample flows toward the first and second ends;(d) conducting the diluted sample from one portion of the second porousmembrane into a first flow path that provides flow from the secondporous membrane upstream to an absorbent pad downstream, wherein theflow path comprises porous membranes and reagents to evaluate reagentreactivity comprising in flow order: (i) a membrane containingmobilizable marker for the class of analytes being detected, (ii) abridging membrane containing mobilizable binding partners specific todownstream immobilized ligands being used to detect the analytes ofinterest in the diluted sample, the mobilized binding partners capableof being recognized by the mobilizable marker, (iii) a membranecontaining immobilized ligands for the analytes of interest, and (iv) anabsorbent pad either in direct fluid communication with the membrane forimmobilized ligands or connected thereto via an outflow wick membrane;(e) conducting the diluted sample from different portions of the secondporous membrane into a second flow path that provides flow from thesecond porous membrane upstream to an absorbent pad downstream andcomprises porous membranes and reagents to detect specific analytes ofinterest comprising in flow order: (i) a membrane containing themobilizable marker, (ii) a bridging membrane connecting the mobilizablemarker membrane to a membrane containing immobilized ligands fordetecting specific analytes of interest, and (iii) an absorbent padeither in direct fluid communication with the membrane for immobilizedligands or connected to it via an outflow wick membrane; (f) observingthe results of migration of the liquid sample through the first flowpath comprising observable marker bound to immobilized ligand or ligandsfor the analyte or analytes of interest, which allows confirmation thatthe marker was reactive and capable of binding to the class of analytesbeing detected, that the immobilized ligand or ligands were reactive andcapable of binding the specific analytes being detected by the test, andthat the mobilizable binding partners specific for the immobilizedligand or ligands were also reactive, thereby indicating that the testis capable of detecting the analytes of interest in the diluted sample;(g) observing the results of migration of the liquid sample through thesecond flow path comprising observable marker bound to immobilizedligand or ligands for the analyte or analytes of interest, which allowsconfirmation that analytes of interest are present in the dilutedsample, or comprising no observable marker bound to immobilized ligandin the second flow path if no analytes of interest are present in thediluted sample; and (h) observing that no valid interpretation can bereached for the test method if no marker lines are present inimmobilized ligand portion of the first flow path.
 2. The method ofclaim 1, wherein immobilized recombinant protein A is immobilizeddownstream from immobilized antigen on one of the flow paths, and usedto evaluate whether sufficient immunoglobulin is present in the dilutedtest sample to allow detection of antibodies specific to that antigen inthe diluted test sample.
 3. The method of claim 1, wherein themobilizable marker is selected from the group consisting of protein G,protein L and protein A, each bound to one or more observable componentsselected from the group consisting of colloidal gold, magnetic spheresand fluorescent markers.
 4. The method of claim 1, wherein themobilizable marker is protein G and protein L bound to colloidal gold.5. The method of claim 1, wherein the membrane containing mobilizablemarker delivers marker to the second flow path during 50% or more of thetotal time period for assay completion.
 6. The method of claim 1,wherein the membrane containing mobilizable marker delivers marker tothe second flow path during more than 75% of the total time period forassay completion.
 7. The method of claim 1, wherein the diluted samplesecond porous membrane is aligned with the mobilizable marker membranecontaining mobilizable marker in the second flow path to provide markermixing with a range of dilutions of diluted sample, from highestconcentration of diluted sample to dilutions containing only 20 percentor less of the highest concentration of diluted sample.
 8. The method ofclaim 1, wherein the liquid sample is a biologic sample from a mammal.9. The method of claim 1, wherein the liquid sample is a biologic samplefrom a non-mammal.
 10. The method of claim 1, wherein the liquid sampleis whole blood or a fraction thereof.
 11. The method of claim 1, whereinthe liquid sample is gingival crevice oral fluid.
 12. The method ofclaim 1, wherein the analyte binding ligand is the antigen and theanalyte is the antibody.
 13. The method of claim 1, wherein the analytebinding ligand is the antibody and the analyte is the antigen.
 14. Themethod of claim 1, wherein the analytes are the antibodies to HIV andthe analyte binding ligands are HIV antigens.
 15. The method of claim 1,wherein the sample diluent comprises a fluid that provides favorable pHand ionic conditions for effective reactions between analytes and theirbinding partners, and contains added protein and detergent to promotemigration of reagents without non-specific adherence of components alongthe separate flow paths.
 16. The method of claim 1, wherein theabsorbent pad has a capacity to receive and hold the total liquid volumeadded to the test device.
 17. A device for detecting an analyte in aliquid sample and checking the reactivity of the reagents used to detectthe analyte, comprising: (a) a compressible porous membrane forreceiving a liquid sample; (b) first and second members adjacentopposing major surfaces of the compressible porous membrane, wherein thefirst and second members are engageable to apply compressive force ontop and bottom surfaces of the membrane along a perimeter of the area tobe isolated, thereby defining a non-compressed area of membranecentripetal to the compressed perimeter containing at least a portion ofa liquid sample to be analyzed; (c) a port to receive delivery of asecond fluid, wherein the port is delivers the second fluid to the topsurface of the isolated portion of the membrane to force the removal ofat least a portion of the liquid sample from the isolated portion of theporous sample receiving membrane; (d) a channel to collect the dilutedliquid sample from the bottom surface of the isolated portion ofmembrane; (e) a second porous membrane for receiving the diluted samplefrom the isolated compressible sample membrane, the second porousmembrane having first and second ends, and where the diluted samplemigrates from point of application to each end of the second porousmembrane; (f) a first flow path for evaluating reagent reactivity thatdelivers diluted sample from one portion of the second porous membraneto an absorbent pad downstream and comprises porous membranes andreagents to evaluate reagent reactivity comprising in flow order: (i) amembrane containing mobilizable marker for the class of analytes beingdetected, (ii) a bridging membrane containing mobilizable bindingpartners specific to downstream immobilized ligands being used to detectthe analytes of interest in the diluted sample, the mobilized bindingpartners capable of being recognized by the mobilizable marker, (iii) amembrane containing immobilized ligands for the analytes of interest,and (iv) absorbent pad positioned at a pre-defined location in thedevice; and (g) a second flow path for detecting specific analytes thatdelivers diluted sample from other portions of the second porousmembrane to an absorbent pad downstream and comprises porous membranesand reagents to detect specific analytes of interest comprising in floworder: (i) a membrane containing the mobilizable marker, (ii) a bridgingmembrane connecting the mobilizable marker membrane to a membranecontaining immobilized ligands for detecting specific analytes ofinterest, and (iii) an absorbent pad held within a defined location ofthe device.
 18. The device of claim 17, wherein the compressible porousmembrane is a hydrophilic glass membrane with low protein binding thatretards cellular components from non-cellular components of whole bloodduring lateral migration through the membrane.
 19. The device of claim18, wherein the compressible porous membrane positioned to collect wholeblood on one end, allow free migration along the membrane to an openingprovided at the opposing end of the insert, and that allows isolation,removal and delivery of the acellular enriched components of whole bloodto the test device.
 20. The device of claim 17, wherein the compressibleporous membrane for use with gingival crevice or other oral fluid is ahydrophilic membrane that saturates with oral fluid, is sonically weldedto a close fit around a swab for collecting oral fluid, and releases theoral fluid sample into the device with minimal protein loss.
 21. Thedevice of claim 17, wherein a swab surrounded on its oral fluidcollection end by the membrane fits into an opening of the device thatallows isolation, removal and delivery of diluted oral fluid into thetest device.
 22. The device of claim 17, wherein the porous dilutedsample membrane comprises a hydrophilic large pore glass membrane withsufficient binder to prevent migration of small glass particles from themembrane into the reagent reactivity and specific antibody flow pathsthat originate from it.
 23. The device of claim 17, wherein the membranecontaining the mobilizable marker of both flow paths comprises ahydrophilic large pore glass or polyester membrane modified to behydrophilic, such that adherence of marker complex to the membrane andrestriction of flow of the mobilizable marker complex from the membraneis minimized when marker complex is dried onto the membrane in thepresence of sucrose or other sugars, and mobilized from the dried statewith liquid sample diluent that promotes migration with minimaladherence of reagents along the flow paths.
 24. The device of claim 17,wherein the bridging membrane of both flow paths comprises a large poreglass with minimal protein binding.
 25. The device of claim 17, whereinthe bridging membrane in the first flow path comprises binding partnersspecific to downstream immobilized ligands dried in a mobilizable state.26. The device of claim 17, wherein the bridging membrane in the secondflow path does not comprise analytes specific to downstream immobilizedligands.
 27. The device of claim 17, wherein the absorbent pad has acapacity to receive and hold the total liquid volume added to thedevice.
 28. The device of claim 17, further comprising apertures,through which one may observe the results of binding or no binding ofmarker to ligands immobilized on the ligand binding membrane of bothflow paths.
 29. A kit for detecting antibodies to HIV in whole blood orcomponents thereof, comprising: (a) a lancet for obtaining fingerstickblood; (b) an insert for collecting blood drops and for inserting intothe device of claim 16; (c) the device of claim 17; and (d) a vial orsyringe to deliver a predetermined amount of sample diluent underpressure through a port of the device.
 30. A kit for detectingantibodies to HIV in gingival crevice oral fluid, comprising: (a) a swabfor collecting oral fluid and for inserting into the device of claim 16;(b) the device of claim 17; and (c) a vial or syringe to deliver apredetermined amount of sample diluent through a port of the device.